Methods and compositions relating to hematopoietic stem cell expansion, enrichment, and maintenance

ABSTRACT

The methods and compositions described herein relate to producing, expanding, enriching, and/or maintaining hematopoietic stem cells ex vivo by treating the cells with an agent(s) that exhibits two or more activities selected from modulation of histone methylation; inhibition of TGFβ signaling; inhibition of p38 signaling; activation of canonical Wnt signaling; and modulation of histone acetylation. In some embodiments, the technology described herein relates to transplantation of hematopoietic stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/US2016/039303 filed Jun. 24, 2016,which designates the U.S. and claims benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 62/184,599 filed Jun. 25, 2015 and62/207,136 filed Aug. 19, 2015, the contents of which are incorporatedherein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 13, 2016, isnamed 701039-084082-PCT_SL.txt and is 3,946 bytes in size.

TECHNICAL FIELD

The technology described herein relates to compositions and methods forthe ex vivo expansion, enrichment, and maintenance of hematopoietic stemcells.

BACKGROUND

While hematopoietic stem cells have significant therapeutic potential, alimitation that has hindered their use in the clinic has been thedifficulty associated with obtaining sufficient numbers of these cells.In particular, hematopoietic stem cells are resistant to maintenance,propagation, and expansion ex vivo. Another challenge to be overcome inorder to further develop the use of hematopoietic stem cells (HSCs) as atherapeutic modality is the loss of multi-potency that can occur whenthese cells are cultured ex vivo. There is currently a need forcompositions and methods for the ex vivo maintenance, propagation, andexpansion of HSCs that preserve the multi-potency and hematopoieticfunctionality of these cells. Described herein is the inventors'discovery of a number of compounds that permit hematopoietic stem cellex vivo maintenance, propagation, expansion and enrichment. Thecompositions and methods of the invention address the challenges posedby conventional HSC therapies by providing strategies for maintaining,propagating and expanding these cells and also enriching heterogeneouscell populations with HSCs while preserving the ability of the ex vivocultured cells to self-renew and differentiate into a multitude ofdifferent blood cell types both in vitro and upon transplantation into arecipient.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods for theexpansion, enrichment, and maintenance of hematopoietic stem cellsduring ex vivo culturing. The compositions and methods of the inventioncan be used to produce expanded populations of hematopoietic stem cellsthat have retained the ability to differentiate into a multitude of celltypes within the hematopoietic lineage. The invention additionallyprovides methods for introducing and modulating the expression ofpolynucleotides within hematopoietic stem cells during ex vivopropagation. Hematopoietic stem cells or progeny thereof producedaccording to the compositions and methods of the invention or cellsderived from these hematopoietic stem cells can be infused into arecipient, such as a human patient, in order to treat a variety ofpathologies. The present invention also provides media for culturinghematopoietic stem cells that contain agents useful for the expansion,enrichment, and maintenance of these cells. Additionally, the inventionprovides kits containing compositions of the invention described herein.

In a first aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more agents that together exhibit one or more activities selectedfrom the group consisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

such that the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells. In some embodiments, the one or more agents together exhibit twoor more activities selected from the above group.

In a second aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with one or more agents thattogether exhibit one or more activities selected from the groupconsisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

such that the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells. In some embodiments, the one or more agents together exhibittwo or more activities selected from the above group.

In an additional aspect, the invention relates to a method ofmaintaining the hematopoietic stem cell functional potential of apopulation of hematopoietic stem cells ex vivo for at least two days bycontacting a first population of hematopoietic stem cells with one ormore agents that together exhibit one or more activities selected fromthe group consisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

such that the population of hematopoietic stem cells contacted with theagents exhibit a hematopoietic stem cell functional potential after twoor more days that is greater than that of a control population ofhematopoietic stem cells cultured under the same conditions and for thesame time as the first population of hematopoietic stem cells but notcontacted with the one or more agents. In some embodiments, the one ormore agents together exhibit two or more activities selected from theabove group.

In embodiments of the above aspects of the invention, the modulation ofhistone methylation is activation of histone methylation, maintenance ofhistone methylation, or inhibition of histone demethylation. Inadditional embodiments, the modulation of histone acetylation isactivation of histone acetylation, maintenance of histone acetylation,or inhibition of histone deacetylation. In particular embodiments of theinvention, the one or more agents include a compound that activateshistone methylation, maintains histone methylation, or inhibits histonedemethylation and a compound that inhibits TGFβ signaling.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more compounds selected from UM171,structural analogs thereof, and the compounds listed in Table 11. Insome embodiments, the one or more compounds listed in Table 11 includesUM171.

In certain embodiments of the invention, the compound that activateshistone methylation, maintains histone methylation, or inhibits histonedemethylation is a histone demethylase inhibitor and the compound thatinhibits TGFβ signaling is a TGFβ receptor inhibitor. The one or moreagents that inhibit TGFβ signaling may include a TGFβ receptorinhibitor. In some embodiments, the TGFβ receptor inhibitor is selectedfrom the group consisting of ALK5 inhibitor II, LY364947, DMH1, andA83-01. In some embodiments, the TGFβ receptor inhibitor is A83-01.

In particular cases, the histone demethylase inhibitor is a LSD1inhibitor. For instance, the LSD1 inhibitor may be LSD1 inhibitor IVRN-1 or tranylcypromine, and the TGFβ receptor inhibitor may be ALK5inhibitor II (E-616452). In some embodiments, the method includescontacting the population of hematopoietic stem cells with one or moreagents that modulate histone methylation. In some embodiments, the oneor more agents that modulate histone methylation activate histonemethylation, maintain histone methylation, or inhibit histonedemethylation. For instance, the one or more agents that modulatehistone methylation may include a histone demethylase inhibitor. In someembodiments, the histone demethylase inhibitor is a LSD1 inhibitor, suchas a LSD1 inhibitor selected from the group consisting of LSD1 inhibitorIV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1inhibitor III CBB1007, LSD1 inhibitor I, and Tranylcypromine. In someembodiments, the LSD1 inhibitor is Tranylcypromine.

In a further aspect, the present invention provides a method ofproducing an expanded population of hematopoietic stem cells ex vivo bycontacting a population of hematopoietic stem cells with one or moreagents that together inhibit the activity of one or more proteinsselected from the group consisting of:

a. a histone demethylase;

b. a protein that propagates TGFβ signaling;

c. a protein that propagates p38 signaling;

d. a protein that promotes β-catenin degradation; and

e. a histone deacetylase.

such that the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells. In some embodiments, the one or more agents together inhibit theactivity of two or more proteins selected from the above group.

An additional aspect of the invention relates to a method of enriching apopulation of cells with hematopoietic stem cells ex vivo by contactinga population of hematopoietic cells that contains one or morehematopoietic stem cells with one or more agents that together inhibitthe activity of one or more proteins selected from the group consistingof:

a. a histone demethylase;

b. a protein that propagates TGFβ signaling;

c. a protein that propagates p38 signaling;

d. a protein that promotes β-catenin degradation; and

e. a histone deacetylase.

such that the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells. In some embodiments, the one or more agents together inhibitthe activity of two or more proteins selected from the above group.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days by contacting afirst population of hematopoietic stem cells with one or more agentsthat together inhibit the activity of one or more proteins selected fromthe group consisting of:

a. a histone demethylase;

b. a protein that propagates TGFβ signaling;

c. a protein that propagates p38 signaling;

d. a protein that promotes β-catenin degradation; and

e. a histone deacetylase.

such that the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the one ormore agents. In some embodiments, the one or more agents togetherinhibit the activity of two or more proteins selected from the abovegroup.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that modulate histoneacetylation. In some embodiments, the one or more agents that modulatehistone acetylation activate histone acetylation, maintain histoneacetylation, or inhibit histone deacetylation. In some embodiments, theone or more agents that that modulate histone acetylation include ahistone deacetylation inhibitor, such as a histone deacetylase inhibitorselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax. In some embodiments, the histonedeacetylase inhibitor is Trichostatin A.

In certain embodiments of the above aspects of the invention, the one ormore agents include a combination of agents selected from thecombination of agents of Table 1, Table 2, Table 3, Table 4, Table 5,and Table 6.

In particular embodiments of the above aspects of the invention, thehistone demethylase is LSD1. In additional embodiments, the one or moreagents include a histone demethylase inhibitor selected from the groupconsisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine. In some cases, the protein that propagates TGFβsignaling is a TGFβ receptor. In additional embodiments, the one or moreagents include a compound that inhibits a protein that propagates TGFβsignaling selected from the group consisting of ALK5 inhibitor II(E-616452), LY364947, A83-01, and DMH1. In certain cases, the one ormore agents include a compound that inhibits a protein that propagatesp38 signaling, and wherein the compound is SB203580. Additionally oralternatively, the one or more agents include a compound that inhibits aprotein that promotes β-catenin degradation selected from the groupconsisting of CHIR99021, lithium chloride, BIO, and FGF2 (also referredto as FGF basic, e.g., recombinant mouse FGF2). In still further cases,the one or more agents include a compound that inhibits a histonedeacetylase are selected from the group consisting of Trichostatin A,valproic acid, butyrylhydroxamic acid, and istodax (romidepsin). Inother embodiments, the one or more agents together inhibit the activityof a histone demethylase and a protein that propagates TGFβ signaling.In certain embodiments, the histone demethylase is LSD1. In additionalembodiments, the protein that propagates TGFβ signaling is a TGFβreceptor. In particular cases, the one or more agents include LSD1inhibitor IV RN-1 and ALK5 inhibitor II (E-616452). In furtherembodiments, the one or more agents include a compound that inhibits p38signaling. In still other cases, the one or more agents include acompound that inhibits a histone deacetylase. In other embodiments, theone or more agents further include a compound that inhibits BMPsignaling. In certain embodiments, the one or more agents include acombination of inhibitors or other agents specified in any one of Tables1-10.

In an additional aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo by contacting apopulation of hematopoietic stem cells with (a) a first agent selectedfrom the group consisting of an LSD1 inhibitor IV RN-1, LSD1 inhibitorII S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1inhibitor I, and Tranylcypromine, and (b) a second agent selected fromthe group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01,Trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax(romidepsin).

In another aspect, the invention relates to a method of enriching apopulation of cells with hematopoietic stem cells ex vivo by contactinga population of hematopoietic cells that contains one or morehematopoietic stem cells with (a) a first agent selected from the groupconsisting of an LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine, and (b) a second agent selected from the groupconsisting of ALK5 inhibitor II (E-616452), LY364947, A83-01,Trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax(romidepsin).

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days by contacting afirst population of hematopoietic stem cells with (a) a first agentselected from the group consisting of LSD1 inhibitor IV RN-1, LSD1inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007,LSD1 inhibitor I, and Tranylcypromine, and (b) a second agent selectedfrom the group consisting of ALK5 inhibitor II (E-616452), LY364947,A83-01, Trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, andistodax (romidepsin), wherein the first population of hematopoietic stemcells exhibits a hematopoietic stem cell functional potential after twoor more days that is greater than that of a control population ofhematopoietic stem cells cultured under the same conditions and for thesame time as the first population of hematopoietic stem cells but notcontacted with the first and second agents.

In embodiments of any of the above-described aspects of the invention,the one or more agents may be a combination of agents selected from thecombination of agents of Table 7, Table 8, Table 9, and Table 10. Incertain embodiments of the above aspects of the invention, the one ormore agents are present in amounts that are sufficient to stimulateexpansion of the population of hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells not contacted withthe one or more agents after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).In additional embodiments, the one or more agents are present in amountsthat are sufficient to stimulate expansion of the population ofhematopoietic stem cells by 10% or more relative to a population ofhematopoietic stem cells that have been contacted with a substance thatinhibits aryl hydrocarbon receptor signaling (such as StemRegenin 1,also referred to as SR1, or an analog thereof), UM171 or an analogthereof, a prostaglandin, an agonist of Notch signaling, or an inhibitorof SIRT1, such as nicotinamide, cambinol, or an analog thereof, afterseven or more days of culture (e.g., after seven, ten, twelve, fourteen,fifteen, twenty, or more days of culture). In certain cases, the one ormore agents are present in amounts that are sufficient to enrich thepopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells not contacted withthe one or more agents after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).In still other embodiments, the one or more agents are present inamounts that are sufficient to enrich the population of cells withhematopoietic stem cells by 10% or more relative to a population ofhematopoietic stem cells that have been contacted with a substance thatinhibits aryl hydrocarbon receptor signaling (such as SR1 or an analogthereof), UM171 or an analog thereof, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1, such as nicotinamide,cambinol, or an analog thereof, after seven or more days of culture(e.g., after seven, ten, twelve, fourteen, fifteen, twenty, or more daysof culture).

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more compounds selected from UM171,structural analogs thereof, and the compounds listed in Table 11. Insome embodiments, the one or more compounds listed in Table 11 includesUM171.

In some embodiments, the method further includes contacting thepopulation of hematopoietic stem cells with one or more agents thatinhibit TGFβ signaling. The one or more agents that inhibit TGFβsignaling may include a TGFβ receptor inhibitor. In some embodiments,the TGFβ receptor inhibitor is selected from the group consisting ofALK5 inhibitor II, LY364947, DMH1, and A83-01. In some embodiments, theTGFβ receptor inhibitor is A83-01.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that modulate histonemethylation. In some embodiments, the one or more agents that modulatehistone methylation activate histone methylation, maintain histonemethylation, or inhibit histone demethylation. For instance, the one ormore agents that modulate histone methylation may include a histonedemethylase inhibitor. In some embodiments, the histone demethylaseinhibitor is a LSD1 inhibitor, such as a LSD1 inhibitor selected fromthe group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I,and Tranylcypromine. In some embodiments, the LSD1 inhibitor isTranylcypromine.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that modulate histoneacetylation. In some embodiments, the one or more agents that modulatehistone acetylation activate histone acetylation, maintain histoneacetylation, or inhibit histone deacetylation. In some embodiments, theone or more agents that that modulate histone acetylation include ahistone deacetylation inhibitor, such as a histone deacetylase inhibitorselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax. In some embodiments, the histonedeacetylase inhibitor is Trichostatin A.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that exhibit one ormore activities selected from the group consisting of:

-   -   a. inhibition of p38 signaling; and    -   b. activation of canonical Wnt signaling.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that inhibit arylhydrocarbon receptor signaling, such as SR1.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more compounds listed in Table 11 and one or more agents that exhibitone or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor signaling,

wherein the one or more compounds and one or more agents are present inamounts that are sufficient to produce an expanded population ofhematopoietic stem cells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with one or more compounds listedin Table 11 and one or more agents that exhibit one or more activitiesselected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor signaling,

wherein the one or more compounds and one or more agents are present inamounts that are sufficient to produce a population of cells enrichedwith hematopoietic stem cells.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withone or more compounds listed in Table 11 and one or more agents thatexhibit one or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor signaling,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the one ormore compounds and one or more agents.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more agents that inhibit aryl hydrocarbon receptor signaling and oneor more agents that exhibit one or more activities selected from thegroup consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with one or more agents thatinhibit aryl hydrocarbon receptor signaling and one or more agents thatexhibit one or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withone or more agents that inhibit aryl hydrocarbon receptor signaling andone or more agents that exhibit one or more activities selected from thegroup consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the one ormore agents.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more compounds selected from UM171,structural analogs thereof, and the compounds listed in Table 11.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a TGFβ receptor inhibitor selected fromthe group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a histone demethylase inhibitor selectedfrom the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor IIS2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1inhibitor I, and Tranylcypromine.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a histone deacetylase inhibitor selectedfrom the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents selected from the groupconsisting of:

-   -   a. a compound that inhibits a protein that propagates p38        signaling including SB203580;

and

-   -   b. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more agents that inhibit arylhydrocarbon receptor, such as SR1.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a compound that inhibits BMP signaling.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more compounds listed in Table 11 and one or more agents that inhibitthe activity of one or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor,

wherein the one or more compounds and one or more agents are present inamounts that are sufficient to produce an expanded population ofhematopoietic stem cells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with one or more compounds listedin Table 11 and one or more agents that inhibit the activity of one ormore proteins selected from the group consisting of:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor,

wherein the one or more compounds and one or more agents are present inamounts that are sufficient to produce a population of cells enrichedwith hematopoietic stem cells.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withone or more compounds listed in Table 11 and one or more agents thatinhibit the activity of one or more proteins selected from the groupconsisting of:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the one ormore compounds and one or more agents.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more agents that inhibit aryl hydrocarbon receptor and one or moreagents that inhibit the activity of one or more proteins selected fromthe group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with one or more agents thatinhibit aryl hydrocarbon receptor and one or more agents that inhibitthe activity of one or more proteins selected from the group consistingof:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withone or more agents that inhibit aryl hydrocarbon receptor and one ormore agents that inhibit the activity of one or more proteins selectedfrom the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the one ormore agents.

In some embodiments, the method further includes contacting thepopulation of hematopoietic stem cells with one or more compoundsselected from UM171, structural analogs thereof, and the compoundslisted in Table 11.

In some embodiments, the method further including contacting thepopulation of hematopoietic stem cells with a TGFβ receptor inhibitorselected from the group consisting of ALK5 inhibitor II, LY364947, DMH1,and A83-01.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a histone demethylase inhibitor selectedfrom the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor IIS2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1inhibitor I, and Tranylcypromine.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with a histone deacetylase inhibitor selectedfrom the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with one or more compounds selected from thegroup consisting of:

-   -   a. a compound that inhibits a protein that propagates p38        signaling including SB203580;    -   and    -   b. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2.

In some embodiments, the method includes contacting the population ofhematopoietic stem cells with SR1.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with oneor more compounds selected from UM171, structural analogs thereof, andthe compounds listed in Table 11; and one or more agents selected fromthe group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2;    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax; and    -   f. an aryl hydrocarbon receptor inhibitor comprising SR1,

wherein the one or more compounds selected from UM171, structuralanalogs thereof, and the compounds listed in Table 11 and the one ormore agents are present in amounts that are sufficient to produce anexpanded population of hematopoietic stem cells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with UM171 and one or more agentsselected from the group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2;    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax; and    -   f. an aryl hydrocarbon receptor inhibitor comprising SR1,

wherein the UM171 and the one or more agents are present in amounts thatare sufficient to produce a population of cells enriched withhematopoietic stem cells.

In another aspect, the invention provides method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withone or more compounds selected from UM171, structural analogs thereof,and the compounds listed in Table 11 and one or more agents selectedfrom the group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2;    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax; and    -   f. an aryl hydrocarbon receptor inhibitor comprising SR1,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the UM171and the one or more agents.

In another aspect, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic stem cells with SR1and one or more agents selected from the group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2; and    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax,

wherein the SR1 and the one or more agents are present in amounts thatare sufficient to produce an expanded population of hematopoietic stemcells.

In another aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo, the methodincluding contacting a population of hematopoietic cells that containsone or more hematopoietic stem cells with SR1 and one or more agentsselected from the group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2; and    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax,

wherein the SR1 and the one or more agents are present in amounts thatare sufficient to produce a population of cells enriched withhematopoietic stem cells.

In another aspect, the invention provides a method of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells ex vivo for at least two days, the methodincluding contacting a first population of hematopoietic stem cells withSR1 and one or more agents selected from the group consisting of:

-   -   a. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a compound that inhibits a protein that propagates p38        signaling comprising SB203580;    -   d. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2; and    -   e. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as the firstpopulation of hematopoietic stem cells but not contacted with the SR1and the one or more agents.

In some embodiments, the one or more agents or compounds are present inamounts that are sufficient to stimulate expansion of the population ofcells by 10% or more relative to a population of hematopoietic stemcells not contacted with the one or more agents or compounds after sevenor more days of culture (e.g., after seven, ten, twelve, fourteen,fifteen, twenty, or more days of culture).

In some embodiments, the one or more agents or compounds are present inamounts that are sufficient to stimulate expansion of the population ofcells by 10% or more relative to a population of hematopoietic stemcells that have been contacted with a prostaglandin, an agonist of Notchsignaling, or an inhibitor of SIRT1 such as nicotinamide, cambinol, oran analog thereof, after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).

In some embodiments, the one or more agents or compounds are present inamounts that are sufficient to enrich the population of cells withhematopoietic stem cells by 10% or more relative to a population ofhematopoietic stem cells not contacted with the one or more agents orcompounds after seven or more days of culture (e.g., after seven, ten,twelve, fourteen, fifteen, twenty, or more days of culture).

In some embodiments, the one or more agents or compounds are present inamounts that are sufficient to enrich the population of cells withhematopoietic stem cells by 10% or more relative to a population ofhematopoietic stem cells that have been contacted with a prostaglandin,an agonist of Notch signaling, or an inhibitor of SIRT1 such asnicotinamide, cambinol, or an analog thereof, after seven or more daysof culture (e.g., after seven, ten, twelve, fourteen, fifteen, twenty,or more days of culture).

In some embodiments, the first population of hematopoietic stem cellsexhibits a hematopoietic stem cell functional potential after three ormore days of culture (e.g., three, five, seven, ten, twelve, fourteen,fifteen, twenty, or more days) that is greater than that of the controlpopulation of hematopoietic stem cells.

In some embodiments, the hematopoietic stem cells are mammalian cells,such as human cells. In some embodiments, the hematopoietic stem cellsare CD34+ cells. In some embodiments, at least 10% of the CD34+ cellsare CD34+, CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+ cells. In some embodiments, thehematopoietic stem cells are from human cord blood. In some embodiments,the hematopoietic stem cells are from human mobilized peripheral blood.In some embodiments, the hematopoietic stem cells are from human bonemarrow. In some embodiments, the hematopoietic stem cells are freshlyisolated from the human. In some embodiments, the hematopoietic stemcells have been previously cryopreserved. In some embodiments, themammalian cells are murine cells.

In embodiments of the above-described methods of maintaining thehematopoietic stem cell functional potential of a population ofhematopoietic stem cells, the population of hematopoietic stem cellsexhibits a hematopoietic stem cell functional potential after two ormore days of culture (e.g., three, five, seven, ten, twelve, fourteen,fifteen, twenty, or more days) that is greater than that of the controlpopulation of hematopoietic stem cells.

In embodiments of any of the above-described aspects of the invention,the hematopoietic stem cells are mammalian cells. In certain cases, themammalian cells are human cells. In particular embodiments, the humancells are CD34+ cells. In certain cases, at least 10% of the CD34+ cellsare CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+ cells. In certain embodiments, thehematopoietic stem cells are from human cord blood. In otherembodiments, hematopoietic stem cells are from human mobilizedperipheral blood. In alternative embodiments, the hematopoietic stemcells are from human bone marrow. In certain cases, the hematopoieticstem cells are freshly isolated from the human. In other cases, thehematopoietic stem cells have been previously cryopreserved. Inparticular embodiments of the invention, the mammalian cells are murinecells.

In further embodiments of any of the above-described methods of theinvention, the hematopoietic stem cells are cultured for two or moredays (e.g., three, five, seven, ten, twelve, fourteen, fifteen, twentyor more days). In certain cases, the hematopoietic stem cells contactthe one or more agents for two or more days (e.g., three, five, seven,ten, twelve, fourteen, fifteen, twenty, or more days). In some cases,the hematopoietic stem cells are contacted with the one or more agentssimultaneously. In other embodiments, the hematopoietic stem cells arecontacted with the one or more agents at different times.

In certain embodiments of the above-described methods of the invention,the hematopoietic stem cells maintain hematopoietic stem cell functionalpotential after two days (e.g., three, five, seven, ten, twelve,fourteen, fifteen, twenty, or more days) in culture. In particularcases, the hematopoietic stem cells maintain hematopoietic stem cellfunctional potential following transplantation after two or more days(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty ormore days) in culture. In other embodiments, the hematopoietic stemcells maintain long term engraftment potential after two or more days(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty, ormore days) in culture. In certain cases, upon transplantation into apatient, the hematopoietic stem cells give rise to recovery of apopulation of cells selected from the group consisting of neutrophils,platelets, red blood cells, monocytes, macrophages, antigen-presentingcells, microglia, osteoclasts, dendritic cells, and lymphocytes. Inparticular embodiments, the lymphocytes are selected from the groupconsisting of natural killer (NK) cells, T cells (e.g., CD4+ or CD8+cells), and B cells. In certain cases, the hematopoietic stem cells arecapable of localizing to hematopoietic tissue to reestablish productivehematopoiesis in a transplanted recipient.

In additional embodiments of the invention, the hematopoietic stem cellsare cultured on a plastic surface or on a substrate includingvitronectin, fibronectin, or matrigel. In certain cases, thehematopoietic stem cells are cultured in the presence of 2-20% oxygen,2-12% oxygen, or about 5% oxygen. In some embodiments, the hematopoieticstem cells are cultured in the presence of 2-12% oxygen. In someembodiments, the hematopoietic stem cells are cultured in the presenceof about 5% oxygen.

In further embodiments of the invention, the hematopoietic stem cellsare originally within a mononuclear cell fraction prior to treatmentwith the one or more agents. In certain cases, the hematopoietic stemcells are originally within a CD34+, CD34+CD38-, CD34+CD38-CD90+,CD34+CD38-CD90+CD45RA−, or CD34+CD38-CD90+CD45RA-CD49F+ enriched cellfraction prior to contacting the one or more agents. In someembodiments, the hematopoietic stem cells are originally within a CD34+,CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+, or CD34+CD38-CD90+CD45RA-CD49F+EPCR+enriched cell fraction prior to contacting the one or more agents orcompounds. In particular cases, the hematopoietic stem cells areoriginally within an un-enriched cell fraction prior to contacting theone or more agents.

In an additional aspect, the invention provides a method of introducingand/or modulating the expression of one or more polynucleotides in apopulation of hematopoietic stem cells, the method including the stepsof:

-   -   a. inserting one or more polynucleotides into the population of        hematopoietic stem cells; and    -   b. expanding or maintaining the population of hematopoietic stem        cells according to the above-described methods of the invention.

In certain cases, (a) precedes (b). In other embodiments, (b) precedes(a).

In another aspect, the invention provides a method of introducing apolynucleotide into a population of hematopoietic stem cells, the methodincluding:

-   -   a. inserting the polynucleotide into the population of        hematopoietic stem cells; and    -   b. expanding the population of hematopoietic stem cells        according to the method of any one of the above embodiments, or        maintaining the hematopoietic stem cell functional potential of        the population of hematopoietic stem cells according to the        method of any one of the above embodiments.

In some embodiments, step (a) above precedes step (b). In someembodiments, step (b) precedes step (a).

In additional embodiments, the method further includes introducing oneor more reagents that cleave a nucleic acid in the cells. In certaincases, the one or more reagents that cleave a nucleic acid in the cellsinclude a zinc finger nuclease (ZFN), a transcription activator-likeeffector nuclease (TALEN), a CRISPR-associated protein, or ameganuclease.

In certain embodiments of the invention, the one or more polynucleotidesare introduced into the hematopoietic stem cells by contacting the cellswith a viral vector (such as a retrovirus, adenovirus (e.g., Ad5, Ad26,Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses),coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g.,influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitisvirus), paramyxovirus (e.g. measles and Sendai), positive strand RNAviruses, such as picornavirus and alphavirus, and double stranded DNAviruses including herpesvirus (e.g., Herpes Simplex virus types 1 and 2,Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia,modified vaccinia Ankara, fowlpox and canarypox)). In other cases, theone or more polynucleotides are introduced into the hematopoietic stemcells by contacting the cells with a vector that encodes a transposableelement (such as the piggybac transposon or the sleeping beautytransposon).

In some embodiments, the method includes contacting the hematopoieticstem cells with a vector selected from the group consisting of a viralvector (such as retrovirus, adenovirus, parvovirus, coronavirus,rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpes virus, orpoxvirus) and a transposable element (such as a piggybac transposon orsleeping beauty transposon).

In certain embodiments of the invention, the one or more polynucleotidesare introduced into the hematopoietic stem cells by electroporation,Nucleofection™, or squeeze-poration.

In some embodiments, the method includes contacting the cells with atransformation agent selected from the group consisting of a cationicpolymer (e.g., diethylaminoethyl-dextran), a cationic lipid, calciumphosphate, an activated dendrimer, and a magnetic bead.

In some embodiments, the method includes introducing the polynucleotideinto the hematopoietic stem cells by microinjection or laserfection.

In some embodiments, the polynucleotide includes a regulatory sequenceselected from the group consisting of a promoter, enhancer, or silencersequence.

In some embodiments, the polynucleotide encodes a molecule selected fromthe group consisting of a protein and a RNA (mRNA, tRNA, siRNA, miRNA,shRNA). In some embodiments, the polynucleotide is a chemically modifiedRNA.

In certain embodiments of the invention, the one or more polynucleotidesare introduced into the hematopoietic stem cells by contacting thehematopoietic stem cells with cationic lipids, cationic polymers such asdiethylaminoethyl (DEAE)-dextran, calcium phosphate, activateddendrimers, magnetic beads, or by microinjection, or laserfection of thehematopoietic stem cells.

In further embodiments, the one or more polynucleotides are introducedinto the hematopoietic stem cells by contacting the cells withnanoparticles including the one or more polynucleotides. In certainembodiments, the one or more polynucleotides are introduced into thehematopoietic stem cells by contacting the cells with one or more VSV-Ginduced microvesicles (also referred to as Gesicles).

In particular cases, the one or more polynucleotides introduced into thepopulation of hematopoietic stem cells contain a gene regulationelement, such as a promoter, enhancer, or silencer. In other cases, theone or more polynucleotides encode a protein or RNA molecule (such asmRNA, tRNA, siRNA, miRNA, or shRNA). In certain embodiments, the one ormore polynucleotides introduced into the population of hematopoieticstem cells are chemically modified RNA molecules.

In additional embodiments, the method further includes introducing intoa recipient the population of expanded hematopoietic stem cells intowhich a polynucleotide has been inserted or progeny thereof.

In another aspect, the invention provides a method of treating arecipient with hematopoietic stem cells or progeny thereof, the methodincluding the steps of:

-   -   a. providing a population of hematopoietic stem cells;    -   b. expanding the population of hematopoietic stem cells        according to the above-described methods of the invention;    -   c. optionally differentiating the hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts,        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of expanded hematopoietic stem        cells or progeny thereof into the recipient.

In an additional aspect, the invention relates to a method of treating arecipient with hematopoietic stem cells or progeny thereof, the methodincluding the steps of:

-   -   a. providing a population of hematopoietic stem cells;    -   b. enriching the population of hematopoietic stem cells        according to the above-described methods of the invention;    -   c. optionally differentiating the hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts,        lymphocytes, NK cells, B-cells and and/or T-cells; and    -   d. introducing the population of cells enriched with        hematopoietic stem cells or progeny thereof into the recipient.

In another aspect, the invention provides a method of treating arecipient with hematopoietic stem cells or progeny thereof, the methodincluding the steps of:

-   -   a. providing a population of hematopoietic stem cells;    -   b. maintaining the hematopoietic stem cell functional potential        of the population of hematopoietic stem cells according to the        above-described methods of the invention;    -   c. optionally differentiating the hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, such as NK cells, B-cells and/or T-cells; and    -   d. introducing the population of hematopoietic stem cells or        progeny thereof into the recipient.

In a further aspect, the invention provides a method of treating arecipient with hematopoietic stem cells or progeny thereof, the methodincluding the steps of:

-   -   a. providing a population of hematopoietic stem cells produced        by the above-described methods of the invention;    -   b. optionally differentiating the hematopoietic stem cells to        into common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts,        lymphocytes, NK cells, B-cells and/or T-cells; and    -   c. introducing the population of hematopoietic stem cells or        progeny thereof into the recipient.

In some embodiments of the above aspects of the invention, the recipientis a human. In particular embodiments, the hematopoietic stem cells arederived from one or more hematopoietic stem cells isolated from a humandonor. In certain cases, the hematopoietic stem cells are from mobilizedperipheral blood of the donor. In additional embodiments, the donor hasbeen previously administered one or more mobilizing agents selected fromthe group consisting of a CXCR4 antagonist (e.g., AMD3100), GCSF, andGROβ.

In some embodiments of any of the above-described methods of theinvention, the hematopoietic stem cells are additionally contacted witha substance that inhibits aryl hydrocarbon receptor signaling. Forinstance, the substance that inhibits aryl hydrocarbon receptorsignaling may be SR1 or an analog thereof. In further embodiments of theinvention, the hematopoietic stem cells are additionally contacted withUM171 or an analog thereof. In still other embodiments, thehematopoietic stem cells are additionally contacted with aprostaglandin, such as dmPGE2 or an analog thereof. In certain cases,the hematopoietic stem cells may be contacted with an agonist of Notchsignaling. In additional embodiments, the hematopoietic stem cells maybe contacted with an inhibitor of SIRT1, such as nicotinamide, cambinol,or an analog thereof.

In some embodiments, the hematopoietic stem cells are additionallycontacted with an agonist of Notch signaling.

In some embodiments, the hematopoietic stem cells are additionallycontacted with an inhibitor of SIRT1. In some embodiments, the inhibitoror SIRT1 is selected from the group consisting of nicotinamide,cambinol, and analogs thereof.

In some embodiments of the methods of the invention, the recipient is ahuman patient suffering from a disease selected from the groupconsisting of Acute Lymphoblastic Leukemia (ALL), Acute MyelogenousLeukemia (AML), Chronic Myelogenous Leukemia (CML), Chronic LymphocyticLeukemia (CLL), Hodgkin Lymphoma (HL), Non-Hodgkin Lymphoma (NHL),Myelodysplastic Syndrome (MDS), Multiple myeloma, Aplastic anemia, Bonemarrow failure, Myeloproliferative disorders such as Myelofibrosis,Essential thrombocytopenia or Polycythemia vera, Fanconi anemia,Dyskeratosis congenita, Common variable immune deficiency (CVID, such asCVID 1, CVID 2, CVID 3, CVID 4, CVID 5, and CVID 6), Humanimmunodeficiency virus (HIV), Hemophagocytic lymphohistiocystosis,Amyloidosis, Solid tumors such as Neuroblastoma, Germ cell tumors,Breast cancer, Wilms' tumor, Medulloblastoma, and Neuroectodermaltumors, Autoimmune diseases such as Scleroderma, Multiple sclerosis,Ulcerative colitis, Systemic lupus erythematosus and Type I diabetes, orprotein deficiencies such as Adrenoleukodystrophy (ALD), Metachromaticleukodystrophy (MLD), Hemophilia A & B, Hurler syndrome, Huntersyndrome, Fabry disease, Gaucher disease, Epidermolysis bullosa, GloboidCell Leukodystrophy, Sanfillipo syndrome, and Morquio syndrome.

In some embodiments, the recipient is a human patient suffering from agenetic blood disease selected from the group consisting of Sickle cellanemia, Alpha thalassemia, Beta thalassemia, Delta thalassemia,Hemoglobin E/thalassemia, Hemoglobin S/thalassemia, HemoglobinC/thalassemia, Hemoglobin D/thalassemia, Chronic granulomatous disease(X-linked Chronic granulomatous disease, autosomal recessive (AR)chronic granulomatous disease, chronic granulomatous disease AR I NCF1,Chronic granulomatous disease AR CYBA, Chronic granulomatous disease ARII NCF2, Chronic granulomatous disease AR III NCF4), X-linked SevereCombined Immune Deficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID,Rag1/Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, Congenitalagranulocytosis, Congenital agranulocytosis-congenital neutropenia-SCN1,Congenital agranulocytosis-congenital neutropenia-SCN2, Familialhemophagocytic lymphohistiocystosis (FHL), Familial hemophagocyticlymphohistiocytosis type 2 (FHL2, perforin mutation), Agammaglobulinemia(X-linked Agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashisyndrome, Hemolytic anemia due to red cell pyruvate kinase deficiency,Paroxysmal nocturnal hemoglobinuria, X-linked Adrenoleukodystrophy(X-ALD), X-linked lymphoproliferative disease, Unicentric Castleman'sDisease, Multicentric Castleman's Disease, Congenital amegakaryocyticthrombocytopenia (CAMT) type I, Reticular dysgenesis, Fanconi anemia,Acquired idiopathic sideroblastic anemia, Systemic mastocytosis, Vonwillebrand disease (VWD), Congenital dyserythropoietic anemia type 2,Cartilage-hair hypoplasia syndrome, Hereditary spherocytosis,Blackfan-Diamond syndrome, Shwachman-Diamond syndrome,Thrombocytopenia-absent radius syndrome, Osteopetrosis, Infantileosteopetrosis, Mucopolysaccharidoses, Lesch-Nyhan syndrome, Glycogenstorage disease, Congenital mastocytosis, Omenn syndrome, X-linkedImmunodysregulation, polyendocrinopathy, and enteropathy (IPEX), IPEXcharacterized by mutations in FOXP3, X-linked syndrome ofpolyendocrinopathy, immune dysfunction, and diarrhea (XPID), X-LinkedAutoimmunity-Allergic Dysregulation Syndrome (XLAAD), IPEX-likesyndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3, HyperIgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin M, Barelymphocyte Syndrome type I, and Bare lymphocyte Syndrome type II (Barelymphocyte Syndrome type II, MHC class I deficiency; Bare lymphocyteSyndrome type II, complementation group A; Bare lymphocyte Syndrome typeII, complementation group C; Bare lymphocyte Syndrome type IIcomplementation group D; Bare lymphocyte Syndrome type II,complementation group E).

In some embodiments, the recipient is a human patient suffering from ahematolymphoid malignancy, a non-hematolymphoid malignancy, or a proteindeficiency, or a tissue or cell transplantation recipient (e.g., toinduce tolerance to transplanted tissue or cells).

Populations of hematopoietic stem cells expanded, enriched, ormaintained by the compositions and/or methods of the invention, as wellas progeny thereof, can also be used to treat a patient (e.g., a humanpatient) suffering from a hematolymphoid malignancy, anon-hematolymphoid malignancy, or a protein deficiency. In otherembodiments, the patient may be the recipient of a tissue or celltransplant, and the hematopoietic stem cells or progeny thereof areadministered in order to induce tolerance to the transplanted tissue orcells.

In some embodiments of the above-described methods of treating a patientwith hematopoietic stem cells or progeny thereof, the hematopoietic stemcells are autologous or syngeneic. Alternatively, the hematopoietic stemcells may be allogeneic.

In an additional aspect, the invention provides a composition includingone or more agents that together exhibit one or more activities selectedfrom the group consisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

In some embodiments, the one or more agents together exhibit two or moreactivities selected from the above group.

In another aspect, the invention provides a composition including:

-   -   a. one or more compounds listed in Table 11; and one or more of    -   b. one or more agents that inhibit TGFβ signaling;    -   c. one or more agents that modulate histone methylation;    -   d. one or more agents that modulate histone acetylation; and    -   e. one or more agents that inhibit aryl hydrocarbon receptor        signaling.

In another aspect, the invention provides a composition including:

-   -   a. one or more agents that inhibit aryl hydrocarbon receptor        signaling; and one or more of    -   b. one or more agents that inhibit TGFβ signaling;    -   c. one or more agents that modulate histone methylation; and    -   d. one or more agents that modulate histone acetylation.

In some embodiments, the compound listed in Table 11 is UM171.

In embodiments, the modulation of histone methylation is activation ofhistone methylation, maintenance of histone methylation, or inhibitionof histone demethylation. In additional embodiments, the modulation ofhistone acetylation is activation of histone acetylation, maintenance ofhistone acetylation, or inhibition of histone deacetylation. Inparticular embodiments, the one or more agents include a compound thatactivates histone methylation, maintains histone methylation, orinhibits histone demethylation and a compound that inhibits TGFβsignaling. In certain cases, the compound that activates histonemethylation, maintains histone methylation, or inhibits histonedemethylation is a histone demethylase inhibitor and the compound thatinhibits TGFβ signaling is a TGFβ receptor inhibitor. In particularembodiments, the histone demethylase inhibitor is a LSD1 inhibitor. Inadditional embodiments, the LSD1 inhibitor is LSD1 inhibitor IV RN-1 andthe TGFβ receptor inhibitor is ALK5 inhibitor II (E-616452).

In some embodiments, the aryl hydrocarbon receptor inhibitor is SR1.

In some embodiments, the composition includes a compound that inhibitsBMP signaling.

In another aspect, the invention provides a composition including one ormore agents that together inhibit the activity of one or more proteinsselected from the group consisting of:

a. a histone demethylase;

b. a protein that propagates TGFβ signaling;

c. a protein that propagates p38 signaling;

d. a protein that promotes β-catenin degradation; and

e. a histone deacetylase.

In some embodiments, the one or more agents together inhibit theactivity of two or more proteins selected from the above group.

In some embodiments of this aspect of the invention, the histonedemethylase is LSD1. In additional embodiments, the one or more agentsinclude a histone demethylase inhibitor selected from the groupconsisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine. In particular cases, the protein that propagates TGFβsignaling is a TGFβ receptor. In some embodiments, the one or moreagents include a compound that inhibits a protein that propagates TGFβsignaling selected from the group consisting of ALK5 inhibitor II(E-616452), LY364947, A83-01, and DMH1. In certain cases, the one ormore agents include a compound that inhibits a protein that propagatesp38 signaling, and wherein the compound is SB203580. In additionalembodiments, the one or more agents include a compound that inhibits aprotein that promotes β-catenin degradation selected from the groupconsisting of CHIR99021, lithium chloride, BIO, and FGF2 (e.g.,recombinant mouse FGF2). In still other embodiments, the one or moreagents include a compound that inhibits a histone deacetylase selectedfrom the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax (romidepsin). In particular cases,the one or more agents together inhibit the activity of a histonedemethylase and a protein that propagates TGFβ signaling. In certainembodiments, the histone demethylase is LSD1. In additional embodiments,the protein that propagates TGFβ signaling is a TGFβ receptor. Incertain cases, the one or more agents include LSD1 inhibitor IV RN-1 andALK5 inhibitor II (E-616452). In additional embodiments, the one or moreagents include a compound that inhibits p38 signaling. In otherembodiments, the one or more agents include a compound that inhibits ahistone deacetylase. In certain cases, the one or more agents include acompound that inhibits BMP signaling.

In another aspect, the invention provides a composition including:

-   -   a. a compound listed in Table 11; and one or more of    -   b. a TGFβ receptor inhibitor;    -   c. a histone demethylase inhibitor;    -   d. a histone deacetylase inhibitor; and    -   e. an aryl hydrocarbon receptor inhibitor.

In another aspect, the invention provides a composition including:

-   -   a. an aryl hydrocarbon receptor inhibitor; and one or more of    -   b. a TGFβ receptor inhibitor;    -   c. a compound listed in Table 11;    -   d. a histone demethylase inhibitor; and    -   e. a histone deacetylase inhibitor.

In some embodiments, the composition includes one or more agentsselected from the group consisting of:

-   -   a. a compound that inhibits a protein that propagates p38        signaling including SB203580;    -   and    -   b. a compound that inhibits a protein that promotes β-catenin        degradation selected from the group consisting of CHIR99021,        lithium chloride, BIO, and FGF2.

In another aspect, the invention provides a composition including:

-   -   a. UM171, a structural analog thereof, or a compound listed in        Table 11; and one or more of    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine;    -   d. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax; and    -   e. SR1.

In another aspect, the invention provides a composition including

-   -   a. SR1; and one or more of    -   b. a TGFβ receptor inhibitor selected from the group consisting        of ALK5 inhibitor II, LY364947, DMH1, and A83-01;    -   c. a histone demethylase inhibitor selected from the group        consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,        LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1        inhibitor I, and Tranylcypromine; and    -   d. a histone deacetylase inhibitor selected from the group        consisting of Trichostatin A, valproic acid, butyrylhydroxamic        acid, and istodax.

In another aspect, the invention provides a composition including (a) afirst agent selected from the group consisting of an LSD1 inhibitor IVRN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitorIII CBB1007, LSD1 inhibitor I, and Tranylcypromine, and (b) a secondagent selected from the group consisting of ALK5 inhibitor II(E-616452), LY364947, A83-01, Trichostatin A, SB203580, CHIR99021, DMH1,sodium acetate, and istodax (romidepsin). In certain cases, the one ormore agents are present in amounts that are sufficient to produce anexpanded population of hematopoietic stem cells. In additionalembodiments, the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells. In still other embodiments, the one or more agents arepresent in amounts sufficient to maintain hematopoietic stem cellfunctional potential of the population of hematopoietic stem cells forat least two days. In particular embodiments, the one or more agents arepresent in an aqueous solution. In other embodiments, the one or moreagents are present as a lyophilized solid.

In some embodiments of the above-described compositions of theinvention, the one or more agents are present in amounts that aresufficient to stimulate expansion of the population of cells by 10% ormore relative to a population of hematopoietic stem cells that have beencontacted with a substance that inhibits aryl hydrocarbon receptorsignaling (such as SR1 or an analog thereof), UM171 or an analogthereof, a prostaglandin, an agonist of Notch signaling, or an inhibitorof SIRT1, such as nicotinamide, cambinol, or an analog thereof, afterseven or more days of culture (e.g., after seven, ten, twelve, fourteen,fifteen, twenty, or more days of culture). In certain cases, the one ormore agents are present in amounts that are sufficient to enrich thepopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells that have beencontacted with a substance that inhibits aryl hydrocarbon receptorsignaling (such as SR1 or an analog thereof), UM171 or an analogthereof, a prostaglandin, an agonist of Notch signaling, or an inhibitorof SIRT1, such as nicotinamide, cambinol, or an analog thereof, afterseven or more days of culture (e.g., after seven, ten, twelve, fourteen,fifteen, twenty, or more days of culture). In particular embodiments,the one or more agents are present in amounts that are sufficient tomaintain long term engraftment potential of the hematopoietic stem cellspost-transplantation after having contacted the cells in culture for twoor more days (e.g., three, five, seven, ten, twelve, fourteen, fifteen,twenty, or more days).

In an additional aspect, the invention provides a composition includinga multi-component combination specified in any one of Tables 1-10.

In another aspect, the invention provides a cell culture mediumincluding any of the above-described compositions of the invention. Incertain cases, the cell culture medium is substantially free of serum.In some embodiments, cytokines may be added to the cell culture media ofthe invention, e.g., in order to further stimulate the proliferation ofhematopoietic stem cells or to induce the differentiation ofhematopoietic stem cells into a desired population of blood cells.

In embodiments of the above-described compositions of the invention, thecomposition may additionally include a population of hematopoietic stemcells in contact with the one or more agents. In certain cases, thehematopoietic stem cells have been cultured in the presence of the oneor more agents for two or more days (e.g., three, five, seven, ten,twelve, fourteen, fifteen, twenty, or more days).

In additional aspects, the invention provides a method of producing anexpanded population of hematopoietic stem cells ex vivo by contacting apopulation of hematopoietic stem cells with one or more agents thattogether exhibit one or more activities selected from the groupconsisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

and wherein the cells are additionally contacted with one or moresubstances selected from the group consisting of a substance thatinhibits aryl hydrocarbon receptor signaling such as SR1 or an analogthereof, UM171 or an analog thereof, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1 such as nicotinamide,cambinol, or an analog thereof, such that the one or more agents and oneor more substances are present in amounts that together are sufficientto produce an expanded population of hematopoietic stem cells.

In a further aspect, the invention provides a method of enriching apopulation of cells with hematopoietic stem cells ex vivo by contactinga population of hematopoietic cells that contains one or morehematopoietic stem cells with one or more agents that together exhibitone or more activities selected from the group consisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

and wherein the cells are additionally contacted with one or moresubstances selected from the group consisting of a substance thatinhibits aryl hydrocarbon receptor signaling such as SR1 or an analogthereof, UM171 or an analog thereof, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1 such as nicotinamide,cambinol, or an analog thereof, such that the one or more agents and oneor more substances are present in amounts that together are sufficientto produce a population of cells enriched with hematopoietic stem cells.

In an additional aspect, the invention relates to a method ofmaintaining the hematopoietic stem cell functional potential of apopulation of hematopoietic stem cells ex vivo for at least two days bycontacting a first population of hematopoietic stem cells with one ormore agents that together exhibit one or more activities selected fromthe group consisting of:

a. modulation of histone methylation;

b. inhibition of TGFβ signaling;

c. inhibition of p38 signaling;

d. activation of canonical Wnt signaling; and

e. modulation of histone acetylation.

wherein the first population of hematopoietic stem cells areadditionally contacted with one or more substances selected from thegroup consisting of a substance that inhibits aryl hydrocarbon receptorsignaling such as SR1 or an analog thereof, UM171 or an analog thereof,a prostaglandin, an agonist of Notch signaling, or an inhibitor of SIRT1such as nicotinamide, cambinol, or an analog thereof, and wherein thefirst population of hematopoietic stem cells exhibits a hematopoieticstem cell functional potential after two or more days that is greaterthan that of a control population of hematopoietic stem cells culturedunder the same conditions and for the same time as the first populationof hematopoietic stem cells but not contacted with the one or moreagents and the one or more substances.

In yet another aspect, the invention provides a population ofhematopoietic stem cells produced by any of the above-described methodsof the invention. In other cases the invention provides a population ofcells derived from the hematopoietic stem cells produced by any of theabove-described methods of the invention.

In an additional aspect, the invention provides a kit including any ofthe above-described compositions of the invention, in addition to apackage insert. In certain cases, the package insert instructs a user ofthe kit to expand, enrich, or maintain a population of hematopoieticstem cells ex vivo. In other cases, the package insert instructs theuser to express a polynucleotide in the hematopoietic stem cells. Inadditional embodiments, the package insert instructs the user toadminister the hematopoietic stem cells to a recipient.

Definitions

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

As used herein, the term “expanded population” of hematopoietic stemcells or hematopoietic progenitor cells refers to a population of cellscomprising at least one more hematopoietic stem cell, such that thequantity of hematopoietic stem cells in the population is greater (e.g.,at least 10% greater, at least 20% greater, at least 30% greater) thanthe number of HSCs prior to administration of one or more agents asdescribed herein (e.g., one or more agents that together exhibit two ormore activities selected from the group consisting of modulation ofhistone methylation, inhibition of TGFβ signaling, inhibition of p38signaling, activation of canonical Wnt signaling, and modulation ofhistone acetylation). In some embodiments, the one or more agentsmodulate TGFβ signaling, modulate lysine methylation, modulate p38signaling, modulate canonical Wnt signaling, modulate histonemethylation, or modulate histone acetylation.

As used herein, an agent that inhibits histone demethylation refers to asubstance or composition (e.g., a small molecule, protein, interferingRNA, messenger RNA, or other natural or synthetic compound, or acomposition such as a virus or other material composed of multiplesubstances) capable of attenuating or preventing the activity of ahistone demethylase or other enzyme that catalyzes the formation of anintermediate that leads to histone demethylation. Inhibition can occurby direct interaction or via indirect means, such as by causing areduction in the quantity of histone demethylase produced in a cell orby inhibition of the interaction between a histone demethylase and amethylated histone substrate. Histone demethylases includelysine-specific demethylases, such as LSD1 and LSD2, as well as otherFAD-dependent histone demethylases. Histone demethylases also includedioxygenases that utilize divalent iron (Fe²⁺) to catalyze oxidativedemethylation of histone residues, such as AlkB, and Jumonji C (JmjC)domain-containing histone demethylases, such as JHDM1, JHDM2, andmembers of the JMJD2 superfamily of histone demethylases. Other enzymesthat convert methylated histone residues into reactive intermediatesthat subsequently undergo oxidative demethylation include monoamineoxidases. Histone demethylation inhibitors may directly bind a histonedemethylase and compete with a methylated histone substrate for bindingat the enzyme active site. Alternatively, an agent that inhibits histonedemethylation may bind a histone demethylase at a location remote fromthe active site and disrupt or prevent the interaction between theenzyme and a methylated histone substrate, e.g., by inducing aconformational change in the enzyme, or disrupt or prevent the catalyticcycle, e.g., by inactivating or displacing enzymatic cofactors.

An agent that inhibits histone demethylation is capable of attenuatingor preventing the formation of a demethylated histone residue with ahalf-maximal inhibitory concentration (IC₅₀) of 100 μM or below (e.g.,between 1 nM and 100 μM) as determined from a histone demethylationassay known in the art or described herein. Exemplary assays that can beused to elucidate the biological activity of a histone demethylationinhibitor include, without limitation, cell-based growth inhibitionassays and dissociation-enhanced lanthanide fluorescence assays asdescribed in U.S. Pat. No. 8,735,622, time-resolved fluorescenceresonance energy transfer assays as described in WO 2014/151945, as wellas mass spectrometry-based assays and coupled-enzyme formaldehydedehydrogenase assays as described in WO 2010/043866, among others.

As used herein, an agent that inhibits the TGFβ signaling pathway refersto a substance or composition (e.g., a small molecule, protein,interfering RNA, messenger RNA, or other natural or synthetic compound,or a composition such as a virus or other material composed of multiplesubstances) that can attenuate or prevent the transcription of one ormore genes that are transcribed due to the activity of a SMADtranscription co-activator protein. An agent that inhibits the TGFβsignaling pathway may disrupt the signal transduction cascade that leadsto SMAD-induced gene transcription at one or more points within thepathway. For instance, a TGFβ signaling pathway inhibitor may disrupt orprevent TGFβ or a TGFβ superfamily ligand, such as Activin, Nodal, bonemorphogenetic protein (BMP), growth and differentiation factor (GDF), orMullerian inhibitory factor (MIF), from binding to its endogenousreceptor, thus inhibiting the phosphorylation and activation of thereceptor-associated SMAD proteins. A TGFβ signaling pathway inhibitormay function by preventing the translocation of one or more SMADproteins to the nucleus, for example, by binding a SMAD protein andpreventing or disrupting the interaction between the SMAD protein andthe nucleoporins. A TGFβ signaling pathway inhibitor may stabilize theinteraction between one or more SMAD proteins and SMAD Anchor forReceptor Activation (SARA), which sequesters SMAD proteins in thecytoplasm and prevents their translocation into the nucleus. Otherexamples of TGFβ signaling pathway inhibitors include substances, suchas neurogenin, that bind SMAD proteins and sequester them from DNA-boundtranscription factors, thus preventing transcription of a target gene.Alternative inhibitors of the TGFβ signaling pathway include substancesthat promote the ubiquitination of one or more SMAD proteins, therebymarking the protein for degradation by the proteasome and preventingtarget gene transcription.

Exemplary assays that can be used to determine the inhibitory activityof a TGFβ signaling pathway inhibitor include, without limitation,electrophoretic mobility shift assays, antibody supershift assays, aswell as TGFβ-inducible gene reporter assays, as described in WO2006/012954, among others.

As used herein, an agent that inhibits the p38 signaling pathway refersto a substance or composition (e.g., a small molecule, protein,interfering RNA, messenger RNA, or other natural or synthetic compound,or a composition such as a virus or other material composed of multiplesubstances) that can attenuate or prevent the activity of the p38mitogen activated protein kinases (MAPKs, e.g., p38α, p38β, p38γ, orp38δ) or any protein that is involved, either directly or indirectly, inactivating one or more of these enzymes. An agent that inhibits the p38signaling pathway may include a substance, such as a monoclonalantibody, that binds to a cytokine receptor, such as IL-1R, and preventsthe receptor-mediated activation of p38 MAP kinases. Alternatively, ap38 signaling pathway inhibitor may bind a p38 protein directly andattenuate or prevent the phosphorylation of the p38 activation loop by aMAP kinase. An agent that inhibits the p38 signaling pathway mayalternatively disrupt the formation of poly-ubiquitin chains at lysineresidues of TNF receptor associated factor (TRAF), which serve asscaffolds for MAPK complexes. Other inhibitors of the p38 signalingpathway include those that promote the phosphorylation of MAP kinases atsites remote from the activation loop and prevent their association withp38, as well as those that acetylate MAP kinases within the activationloop and thus prevent phosphorylation and concomitant activation of theMAP kinase.

Exemplary assays that can be used to determine the inhibitory activityof an agent that inhibits the p38 signaling pathway include, withoutlimitation, fluorescence anisotropy competitive binding assays, as wellas time-resolved fluorescence resonance energy transfer assays, asdescribed in WO 2006/012954, among others.

As used herein, an agent that inhibits histone deacetylation refers to asubstance or composition (e.g., a small molecule, protein, interferingRNA, messenger RNA, or other natural or synthetic compound, or acomposition such as a virus or other material composed of multiplesubstances) capable of attenuating or preventing the activity of histonedeacetylase, more particularly its enzymatic activity either via directinteraction or via indirect means such as by causing a reduction in thequantity of a histone deacetylase produced in a cell or by inhibition ofthe interaction between a histone deacetylase and an acetylated histonesubstrate. Inhibiting histone deacetylase enzymatic activity meansreducing the ability of a histone deacetylase to catalyze the removal ofan acetyl group from a histone residue (e.g., a mono-, di-, ortri-methylated lysine residue; a monomethylated arginine residue, or asymmetric/asymmetric dimethylated arginine residue, within a histoneprotein). Preferably, such inhibition is specific, such that the anagent that inhibits histone deacetylation reduces the ability of ahistone deacetylase to remove an acetyl group from a histone residue ata concentration that is lower than the concentration of the inhibitorthat is required to produce another, unrelated biological effect.

As used herein, the terms “histone deacetylase” and “HDAC” refer to anyone of a family of enzymes that catalyze the removal of acetyl groupsfrom the ε-amino groups of lysine residues at the N-terminus of ahistone. Unless otherwise indicated by context, the term “histone” ismeant to refer to any histone protein, including HI, H2A, H2B, H3, H4,and H5, from any species. Human HDAC proteins or gene products, include,but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6,HDAC-7, HDAC-8, HDAC-9, HDAC-10, and HDAC-11.

As used herein, an agent that inhibits a protein that promotes β-catenindegradation include those agents that inhibit β-catenin phosphorylationor ubiquitination. These agents may be any substance (e.g., a smallmolecule, protein, interfering RNA, messenger RNA, or other natural orsynthetic compound, or a composition such as a virus or other materialcomposed of multiple substances) that can reduce the rate or extent ofβ-catenin degradation, e.g., by attenuating the catalysis ofphosphorylation of serine and/or threonine residues that would otherwiserender β-catenin a substrate for ubiquitination and proteasome-mediateddegradation (for example, at residues Ser33, Ser37 and/or at Thr41). Byextending the half life of functional β-catenin, these agents promote aconcomitant increase in the rate or extent of transcription of a genethat is transcribed due to the activity of the β-catenin transcriptionco-activator. Exemplary agents that inhibit β-catenin phosphorylationinclude agonists of the canonical β-catenin/Wnt signaling pathway, asignal transduction cascade that orchestrates the inhibition of glycogensynthase kinase 3 (GSK3) by providing substrates that compete withβ-catenin for phosphorylation.

As used herein, a “Wnt signaling agonist” refers to an agonist of thecanonical Wnt signaling pathway. Agonists of this pathway furtherinclude Wnt proteins or other compounds that bind directly to theFrizzled and LRP5/6 co-receptor proteins in a manner that promotes anincrease in the concentration of β-catenin in the nucleus of a mammaliancell. Alternatively, a β-catenin/Wnt pathway agonist may function byinhibiting one or more secreted Frizzled-related proteins (SFRPs) or Wntinhibitory protein (WIF), which bind and sequester Wnt proteins from theendogenous Wnt co-receptors.

Exemplary methods that can be used to determine the activity of aβ-catenin/Wnt pathway agonist include, without limitation, monitoringthe expression of a reporter gene under the control of a TCF/LEF familytranscription factor, as well as TOPFlash luciferase reporter assays, asdescribed in US 2014/0044763.

As used herein, a compound that inhibits aryl hydrocarbon receptorsignaling include agents that inhibit the signal transduction cascadethat is propagated by the binding of the aryl hydrocarbon receptor to anactivating ligand thereof. The aryl hydrocarbon receptor is a cytosolic,ligand-inducible transcription factor that, upon binding to an agonisticligand, translocates into the nucleus and promotes the transcription oftarget genes containing the distinct sequence motifs, such as the geneencoding cytochrome P450A1 enzyme that contains an upstreamdioxin-responsive element. Examples of agents that inhibit arylhydrocarbon receptor signaling include aryl hydrocarbon receptorinhibitors, which may include compounds, such as SR1, that bind the arylhydrocarbon receptor directly and thus compete with aryl hydrocarbonreceptor ligands for binding to the receptor. Additional examples ofagents that inhibit aryl hydrocarbon receptor signaling include agentsthat interfere with the translocation of the active aryl hydrocarbonreceptor to the nucleus, and agents that inhibit the interaction of thearyl hydrocarbon receptor with the DNA (e.g., the promoter regionscontaining XRE sites) of target genes.

As used herein, an agonist of Notch signaling is an agent that promotesactivation of Notch pathway function. The term “Notch pathway function”as used herein refers to a function mediated by the Notch signaltransduction pathway including, but not limited to, nucleartranslocation of the intracellular domain of Notch, nucleartranslocation of RBP-Jκ or its Drosophila homolog Suppressor ofHairless; activation of bHLH genes of the Enhancer of Split complex,e.g., Mastermind; activation of the HES-1 gene or the KBF2 (alsoreferred to as CBF1) gene; inhibition of Drosophila neuroblastsegregation; and binding of Notch to a Delta protein, a Jagged/Serrateprotein, Fringe, Deltex or RBP-Jκ/Suppressor of Hairless, or homologs oranalogs thereof. The Notch signal transduction cascade and thephenotypes effected by Notch signaling are described, e.g., in Kopan etal., Cell 137:216 (2009) and Jarriault, et al., Mol. Cell. Biol. 18:7423(1998), the disclosures of each of which are incorporated herein byreference. Examples of Notch agonists are described, e.g., in US2014/0369973 and in U.S. Pat. No. 7,399,633, the disclosures of each ofwhich are incorporated herein by reference. Exemplary Notch agonistsinclude, without limitation, Notch proteins, as well as analogs,derivatives, and fragments thereof; other proteins that propagate theNotch signaling pathway, as well as analogs, derivatives, and fragmentsthereof; activating antibodies that stimulate Notch receptor activityand antigen-binding fragments thereof that retain agonistic activity;nucleic acids encoding proteins that potentiate Notch signaling; as wellas proteins, derivatives, and analogs thereof which bind to or otherwiseinteract with Notch proteins or other proteins in the Notch pathway suchthat Notch pathway activity is promoted. Such agonists include, but arenot limited to, Notch proteins and derivatives thereof containing theNotch intracellular domain, Notch nucleic acids encoding the foregoing,and proteins contacting the Notch-interacting domain of Notch ligands(e.g., the extracellular domain of Delta or Serrate). Other agonistsinclude but are not limited to RBPJκ/Suppressor of Hairless or Deltex.Fringe can additionally be used to enhance Notch activity, for examplein conjunction with Delta protein. These proteins, fragments, andderivatives thereof can be recombinantly expressed and isolated or canbe chemically synthesized using peptide and protein synthesis techniquesknown in the art.

As used herein, the term “inhibitor” refers to any compound, natural orsynthetic, which can reduce the activity of a target protein orsignaling pathway. An inhibitor can be, for example, a peptide, aprotein, an antibody, a peptidomimetic, an amino acid, an amino acidanalog, a polynucleotide, a polynucleotide analog, an aptamer, anucleotide, a nucleotide analog, an organic or inorganic compound. Aninhibitor may attenuate or prevent the activity of a target proteineither directly or indirectly. Direct inhibition can be obtained, forinstance, by binding to a protein and preventing the protein frominteracting with an endogenous molecule, such as an enzyme, a substrate,or other binding partner, thereby diminishing the activity of theprotein. For instance, an inhibitor may bind an enzyme active site andsterically preclude binding of an endogenous substrate at this location,thus decreasing the enzymatic activity of the protein. Alternatively,indirect inhibition can be obtained, for instance, by binding to aprotein that promotes the activity of a target protein by inducing aconformational change or catalyzing a chemical modification of thetarget protein. For instance, indirect inhibition of a target proteinmay be achieved by binding and inactivating a kinase that catalyzes thephosphorylation of, and thus activates, the target protein.

As used herein, the term “hematopoietic stem cells” (or “HSCs”) refer toimmature blood cells having the capacity to self-renew and todifferentiate into mature blood cells comprising diverse lineagesincluding but not limited to granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B-cells and T-cells). It is known in the art that such cells mayor may not include CD34⁺ cells. CD34⁺ cells are immature cells thatexpress the CD34 cell surface marker. In humans, CD34+ cells arebelieved to include a subpopulation of cells with the stem cellproperties defined above, whereas in mice, HSCs are CD34−. In addition,HSCs also refer to long term repopulating HSC (LT-HSC) and short termrepopulating HSC (ST-HSC). LT-HSC and ST-HSC are differentiated, basedon functional potential and on cell surface marker expression. Forexample, human HSC are a CD34+, CD38-, CD45RA−, CD90+, CD49F+, and lin−(negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8,CD10, CD11B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSC areCD34-, SCA-1+, C-kit+, CD135-, Slamfl/CD150+, CD48-, and lin− (negativefor mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8,B220, IL7ra), whereas ST-HSC are CD34+, SCA-1+, C-kit+, CD135-,Slamfl/CD150+, and lin− (negative for mature lineage markers includingTer119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra). In addition, ST-HSC areless quiescent (i.e., more active) and more proliferative than LT-HSCunder homeostatic conditions. However, LT-HSC have greater self renewalpotential (i.e., they survive throughout adulthood, and can be seriallytransplanted through successive recipients), whereas ST-HSC have limitedself renewal (i.e., they survive for only a limited period of time, anddo not possess serial transplantation potential). Any of these HSCs canbe used in any of the methods described herein. Optionally, ST-HSCs areuseful because they are highly proliferative and thus, can more quicklygive rise to differentiated progeny.

As used herein, the phrase “hematopoietic stem cell functionalpotential” refers to the functional properties of hematopoietic stemcells which include 1) multi-potency (which refers to the ability todifferentiate into multiple different blood lineages including, but notlimited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils,basophils), erythrocytes (e.g., reticulocytes, erythrocytes),thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes,platelets), monocytes (e.g., monocytes, macrophages), dendritic cells,microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells andT-cells), 2) self-renewal (which refers to the ability of hematopoieticstem cells to give rise to daughter cells that have equivalent potentialas the mother cell, and further that this ability can repeatedly occurthroughout the lifetime of an individual without exhaustion), and 3) theability of hematopoietic stem cells or progeny thereof to bereintroduced into a transplant recipient whereupon they home to thehematopoietic stem cell niche and re-establish productive and sustainedhematopoiesis.

Hematopoietic stem cells are optionally obtained from blood products. Ablood product includes a product obtained from the body or an organ ofthe body containing cells of hematopoietic origin. Such sources includeunfractionated bone marrow, umbilical cord, placenta, peripheral blood,or mobilized-peripheral blood. All of the aforementioned crude orunfractionated blood products can be enriched for cells havinghematopoietic stem cell characteristics in a number of ways. Forexample, the more mature, differentiated cells are selected against, viacell surface molecules they express. Optionally, the blood product isfractionated by positively selecting for CD34⁺ cells. CD34⁺ cellsinclude a subpopulation of hematopoietic stem cells capable ofself-renewal, multi-potency, and that can be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis. Suchselection is accomplished using, for example, commercially availablemagnetic anti-CD34 beads (Dynal, Lake Success, N.Y.). Unfractionatedblood products are optionally obtained directly from a donor orretrieved from cryopreservative storage. Hematopoietic stem cells canalso be optionally obtained from differentiated embryonic stem cells,differentiated induced pluripotent stem cells or from other reprogrammedmature cells types.

The terms “stem cell” or “undifferentiated cell” as used herein, referto a cell in an undifferentiated or partially differentiated state thathas the property of self-renewal and has the developmental potential todifferentiate into multiple cell types. A stem cell is capable ofproliferation and giving rise to more such stem cells while maintainingits functional potential. Stem cells can divide asymmetrically, which isknown as obligatory asymmetrical differentiation, with one daughter cellretaining the functional potential of the parent stem cell and the otherdaughter cell expressing some distinct other specific function,phenotype and/or developmental potential from the parent cell. Thedaughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. A differentiated cell may derive from amultipotent cell, which itself is derived from a multipotent cell, andso on. Alternatively, some of the stem cells in a population can dividesymmetrically into two stem cells. Accordingly, the term “stem cell”refers to any subset of cells that have the developmental potential,under particular circumstances, to differentiate to a more specializedor differentiated phenotype, and which retain the capacity, undercertain circumstances, to proliferate without substantiallydifferentiating. In some embodiments, the term stem cell refersgenerally to a naturally occurring parent cell whose descendants(progeny cells) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. Cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen can be induced to “reverse” and re-express the stem cell phenotype,a term often referred to as “dedifferentiation” or “reprogramming” or“retrodifferentiation” by persons of ordinary skill in the art.

As used herein, “homing potential” refers to the capability of a stemcell to localize to sites in vivo that can support productivehematopoiesis such as the bone marrow.

As used herein, a cell population that is “enriched” in a particularcell type refers to a population in which the relative proportion ofcells of a particular type has increased in comparison with a previouspopulation of cells (for example, in comparison with a population ofcells prior to treatment with one or more agents that together exhibittwo or more activities selected from the group consisting of modulationof histone methylation, inhibition of TGFβ signaling, inhibition of p38signaling, activation of canonical Wnt signaling, and modulation ofhistone acetylation).

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that has the developmental potential to differentiateinto multiple different hematopoietic cell types. Hematopoietic stemcells are multi-potent and can form the many different types of bloodcells (red, white, platelets, etc.), but it cannot form neurons.

As used herein, the phrases “preserve multi-potency” or “maintainmulti-potency” refer to a process by which the degree of multi-potencyof a population of cells is preserved over a period of time. The degreeof multi-potency of a population of cells describes the number andidentity of differentiated cell types into which a population of cellscan differentiate. For example, a population of cells exhibitingmulti-potency that has been maintained over a period of two days ex vivo(e.g., in culture) is capable of differentiating into at least the samenumber of different cell types as the population was capable ofdifferentiating into at the beginning of the cell culture period.

As used herein, a “mobilizing agent” is an agent capable of inducing themigration of hematopoietic stem cells from the bone marrow of a subjectto the peripheral blood. Exemplary mobilizing agents include CXCR4antagonists, such as AMD3100, as well as GCSF and GROβ.

As used herein, “contacting” a population of cells with one or moreagents can be achieved in a variety of ways. For instance, a populationof hematopoietic stem cells may be contacted with one or more agentsthat together exhibit two or more activities selected from the groupconsisting of modulation of histone methylation, inhibition of TGFβsignaling, inhibition of p38 signaling, activation of canonical Wntsignaling, and modulation of histone acetylation (e.g., LSD1 inhibitorIV RN-1; LSD1 inhibitor II S2101; LSD1 inhibitor LSD1-C76; LSD1inhibitor III CBB1007; LSD1 inhibitor I; ALK5 inhibitor II (E-616452);LY364947; A83-01; Trichostatin A; Tranylcypromine; SB203580; CHIR99021;DMH1; sodium acetate; and istodax) by culturing the hematopoietic stemcells in the presence of these agents for a period of time, such as fortwo or more days. When more than once agent is contacted with apopulation of cells, the agents can be present in the cell culturemedium together, such that the cells are exposed to the one or moreagents simultaneously. Alternatively, the one or more agents may beadded to the cell culture medium sequentially. For instance, the one ormore agents may be added to a population of cells in culture accordingto a particular regimen, e.g., such that different agents are added tothe culture media at different times during a culture period.

As used herein, the term “engraftment potential” is used to refer to theability of hematopoietic stem and progenitor cells to repopulate atissue, whether such cells are naturally circulating or are provided bytransplantation. The term encompasses all events surrounding or leadingup to engraftment, such as tissue homing of cells and colonization ofcells within the tissue of interest. The engraftment efficiency or rateof engraftment can be evaluated or quantified using any clinicallyacceptable parameter as known to those of skill in the art and caninclude, for example, assessment of competitive repopulating units(CRU); incorporation or expression of a marker in tissue(s) into whichstem cells have homed, colonized, or become engrafted; or by evaluationof the progress of a subject through disease progression, survival ofhematopoietic stem and progenitor cells, or survival of a recipient. Inone embodiment, engraftment is determined by measuring white blood cellcounts in peripheral blood during a post-transplant period.Alternatively, engraftment can be assessed by measuring recovery ofmarrow cells by donor cells in a bone marrow aspirate sample.

As used herein, the term “self-renewal” refers to the ability of a stemcell to produce daughter stem cells with the same phenotype,characteristics and functional potential as the original stem cell. Inparticular, self-renewal, as used herein, is defined as the ability tocontinue proliferation while maintaining an undifferentiatedmulti-potent stem cell state.

As used herein, a population of cells that has been “freshly isolated”from a donor refers to a population of cells that has been isolated froma donor without having been cryopreserved and thawed prior to infusion.A population of cells may be isolated from a donor and separated intotwo intermediate populations, one of which may be infused into a patientand the other of which may be cryopreserved. In this instance, theintermediate population that is infused into the patient is consideredfreshly isolated. A population of cells is considered to be freshlyisolated from a donor if the population is cultured ex vivo prior toinfusion in the patient. For example, this culturing step may beperformed in order to expand, enrich, and/or maintain the population ofhematopoietic stem cells prior to administration of the resulting cellsto a patient. In these instances, the resulting cells are considered tobe freshly isolated from the donor, to the extent that the cells thatare administered to the patient have not been cryopreserved and thawedprior to infusion into the patient.

As used herein, the term “ZsGr” refers to the fluorescent cassetteZsGreen, and references to “Fdg5·ZsGr” indicate that the fluorescentZsGreen reporter cassette is knocked in frame into the endogenous Fgd5locus. In these cases, ZsGreen expression is under the control of theFgd5 promoter. The inventors have previously shown that Fgd5 isexclusively expressed in hematopoietic stem cells in the murinehematopoietic system. The inventors have further shown that highly purehematopoietic stem cells can be isolated based on single color ZsGreenfluorescence when using Fdg5·ZsGreen mice. Hematopoietic stem cellsisolated from such mice are referred to as ZsGr+, or Fgd5·ZsGr+. Thisreporter construct is described in further detail in Gazit R, Mandal PK, Ebina W, Ben-Zvi A, Nombela-Arrieta C, Silberstein L E, Rossi D J.Journal of Experimental Medicine. 211(7):1315-31 (2014), the disclosureof which is incorporated herein by reference.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level.

As used herein, “CRU (competitive repopulating unit)” refers to a unitof measure of long-term engrafting stem cells, which can be detectedafter in-vivo transplantation.

As used herein, the term “modulate” means to change or induce analteration in a particular biological activity. Modulation includes, butis not limited to, stimulating or inhibiting an activity (e.g., byactivating a receptor so as to initiate a signal transduction cascade,to inhibit a receptor from propagating a signaling pathway, byactivating an endogenous inhibitor that attenuates a biologicalactivity, or by inhibiting the activity of a protein that inhibits aparticular biological function. Modulation of a protein that propagatesa particular signaling pathway may result in an increase or a decreasein activity (e.g., in histone methylation, TGFβ signaling, p38signaling, Wnt signaling, or histone acetylation), a change in theaffinity of one protein in a pathway for another, or another change inthe structural, functional, or immunological properties associated withthe activity of a pathway or protein within a pathway.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statistically significant amount. Insome embodiments, the terms “increased”, “increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to areference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. In the context of a marker, an “increase” is astatistically significant increase in such level.

As used herein, a “subject” means a human or animal Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, and hamsters. Domestic and game animals includecows, horses, pigs, deer, bison, buffalo, feline species, e.g., domesticcat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken,emu, ostrich, and fish, e.g., trout, catfish and salmon. In someembodiments, the subject is a mammal, e.g., a primate, e.g., a human.The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

As used herein, a “recipient” is a patient that receives a transplant,such as a transplant containing a population of hematopoietic stem cellsor a population of differentiated cells. The transplanted cellsadministered to a recipient may be, e.g., autologous, syngeneic, orallogeneic cells.

As used herein, a “donor” is a human or animal from which one or morecells are isolated prior to administration of the cells, or progenythereof, into a recipient. The one or more cells may be, e.g., apopulation of hematopoietic stem cells to be expanded, enriched, ormaintained according to the methods of the invention prior toadministration of the cells or the progeny thereof into a recipient.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of diseaseand/or treatment. A subject can be male or female.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “polynucleotide”, “nucleic acid” or “nucleicacid sequence” refers to any molecule, preferably a polymeric molecule,incorporating units of ribonucleic acid, deoxyribonucleic acid or ananalog thereof. The nucleic acid can be either single-stranded ordouble-stranded. A single-stranded nucleic acid can be one nucleic acidstrand of a denatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. In another aspect, the nucleic acid can bechemically modified-RNA. For instance, the nucleic acid can bechemically modified messenger RNA. In another aspect, the nucleic acidcan be RNA synthesized with naturally occurring or synthetic nucleotideanalogs. Suitable nucleic acid molecules are DNA, including genomic DNAor cDNA. Other suitable nucleic acid molecules are RNA, including mRNA,tRNA, siRNA, miRNA, or shRNA.

A used herein, the term “siRNA” refers to a double stranded nucleic acidmolecule capable of RNA interference or “RNAi”, as disclosed, forexample, in Bass, Nature 411: 428-429 (2001); Elbashir et al., Nature411: 494-498 (2001); WO 2000/044895; WO 2001/036646; WO 1999/032619; WO2000/001846; WO 2001/029058; WO 1999/007409; and WO 2000/044914, thedisclosures of each of which are incorporated herein by reference. Asused herein, siRNA molecules are not limited to those moleculescontaining only RNA, but further encompass chemically modifiednucleotides and non-nucleotides having RNAi capacity or activity.

As used herein, the term “miRNA” refers to a class of small, non-coding,single-stranded RNA, typically between 18-23 nucleotides in length.miRNA molecules are capable of regulating gene expression by modulatingthe stability and translation of mRNA encoding specific proteins. miRNAalso influence other nuclear processes, such as heterochromatinformation and genome rearrangement.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex containing siRNA, part of which is in the form of a hairpinstructure. In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. For instance, the loop may be5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpinstructure can also contain 3′ or 5′ overhang portions. For example, theoverhang may be a 3′ or a 5′ overhang and may be 0, 1, 2, 3, 4 or 5nucleotides in length.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound, cell, or population of cells as disclosed herein into asubject by a method or route which results in at least partial deliveryof the agent at a desired site. Pharmaceutical compositions comprisingthe compounds or cells disclosed herein can be administered by anyappropriate route which results in an effective treatment in thesubject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” As used herein, the term “about” indicates a deviation of ±10%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,A D A M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

As used herein, “LSD1 inhibitor II S2101” refers to a compound havingthe structure of Formula I or a pharmaceutically acceptable saltthereof. The synthesis of LSD1 inhibitor II S2101 as well as thestructure and synthesis of related compounds, e.g., derivatives,including those with similar biological activity are known in the art,see, e.g. WO 1998/009625; WO 2003/016309; WO 2004/087646; WO2005/033066; WO 2006/034769; WO 2007/068330; WO 2009/147217; WO2014/194280; US 2013/0178520; U.S. Pat. No. 5,426,196; EP 764640; and EP764632, the disclosures of each of which are incorporated herein byreference.

As used herein, “LSD1 inhibitor IV RN-1” refers to a compound having thestructure of Formula II or a pharmaceutically acceptable salt thereof.The synthesis of LSD1 inhibitor IV RN-1 as well as the structure andsynthesis of related compounds, e.g., derivatives, including those withsimilar biological activity are known in the art, see, e.g. WO2015/003643; WO 2014/205266; WO 2014/19428; WO 2012/122405; WO2014/018375; WO 2009/147217; WO 2007/068330; WO 2006/034769; WO2005/033066; WO 2004/087646; WO 2003/016309; WO 1998/009625; WO1997/010822; WO 1997/010825; WO 1995/017408; and US 2013/0178520, eachof which is incorporated by reference herein in its entirety.

As used herein, “LSD1 inhibitor LSD1-C76” refers to a compound havingthe structure of Formula III or a pharmaceutically acceptable saltthereof. The synthesis of LSD1 inhibitor LSD1-C76 as well as thestructure and synthesis of related compounds, e.g., derivatives,including those with similar biological activity are known in the art.

As used herein, “LSD1 inhibitor III CBB1007” refers to a compound havingthe structure of Formula IV or a pharmaceutically acceptable saltthereof. The synthesis of LSD1 inhibitor III CBB1007 as well as thestructure and synthesis of related compounds, e.g., derivatives,including those with similar biological activity are known in the art.

As used herein, “LSD1 inhibitor I” refers to a compound having thestructure of Formula V or a pharmaceutically acceptable salt thereof.LSD1 inhibitor I is also referred to in the art as BHC110 Inhibitor I,Histone Lysine Demethylase Inhibitor III, and/or KDM1 Inhibitor I. Thesynthesis of LSD1 inhibitor I as well as the structure and synthesis ofrelated compounds, e.g., derivatives, including those with similarbiological activity are known in the art.

As used herein, “ALK5 inhibitor II” (also referred to as “RepSox” or“E-616452” refers to a compound having the structure of Formula VI or apharmaceutically acceptable salt thereof. ALK5 inhibitor II (E-616452)is also referred to in the art as Transforming Growth Factor-b Type IReceptor Kinase Inhibitor II or Repsox.

As used herein, “LY364947” refers to a compound having the structure ofFormula VII or a pharmaceutically acceptable salt thereof. LY364947 isalso referred to in the art as ALK5 Inhibitor I TbR-I InhibitorTransforming Growth Factor-b Type I Receptor Kinase Inhibitor. Thesynthesis of LY364947 and ALK5 inhibitor II as well as the structure andsynthesis of related compounds, e.g., derivatives, including those withsimilar biological activity are known in the art, see, e.g. U.S. Pat.No. 6,028,072; WO 2002/062794; WO 2004/026302; WO 2004/026306; WO2004/072033; WO 2007/088651; WO 2007/070866; WO 2007/039151; and WO2007/052943, each of which is incorporated by reference herein in itsentirety.

As used herein, “A83-01” refers to a compound having the structure ofFormula VIII or a pharmaceutically acceptable salt thereof. Thesynthesis of A83-01 as well as the structure and synthesis of relatedcompounds, e.g., derivatives, including those with similar biologicalactivity are known in the art, see, e.g., US 2003/0064997; US2003/0064997; US 2003/0064997; U.S. Pat. Nos. 5,777,097; 5,871,934; GB2306108; WO 1993/014081; WO 1995/003297; WO 1997/33883; WO 1997/35855;and WO 1993/014081; each of which is incorporated by reference herein inits entirety.

As used herein, “trichostatin A” refers to a compound having thestructure of Formula IX or a pharmaceutically acceptable salt thereof.The synthesis of trichostatin A as well as the structure and synthesisof related compounds, e.g., derivatives, including those with similarbiological activity are known in the art, see, e.g. U.S. Pat. Nos.4,690,918; 4,946,999; EP 0827946; JP 07206670; and JP 60149520; each ofwhich is incorporated by reference herein in its entirety.

As used herein, “tranylcypromine” refers to a compound having thestructure of Formula X or a pharmaceutically acceptable salt thereof.The synthesis of tranylcypromine as well as the structure and synthesisof related compounds, e.g., derivatives, including those with similarbiological activity are known in the art, see, e.g. U.S. Pat. Nos.2,993,931; 2,997,422; 3,011,945; 3,079,403; 3,134,676; and 3,244,596,each of which is incorporated by reference herein in its entirety.

As used herein, “SB203580” refers to a compound having the structure ofFormula XI or a pharmaceutically acceptable salt thereof. The synthesisof SB203580 as well as the structure and synthesis of related compounds,e.g., derivatives, including those with similar biological activity areknown in the art, see, e.g., WO 2007/070866; WO 2008/022182; WO2010/065917; WO 2010/077955; and WO 2010/102267; each of which isincorporated by reference herein in its entirety.

As used herein, “CHIR99021” refers to a compound having the structure ofFormula XII or a pharmaceutically acceptable salt thereof. The synthesisof CHIR99021 as well as the structure and synthesis of relatedcompounds, e.g., derivatives, including those with similar biologicalactivity are known in the art, see, e.g. WO 1999/065897; WO 2002/020495;WO 2005/003948; WO 2006/001863; WO 2006/117212; WO 2007/016485; WO2007/075911; WO 2007/083978; and US 2002/0156087; each of which isincorporated by reference herein in its entirety.

As used herein, “DMH1” refers to a compound having the structure ofFormula XIII or a pharmaceutically acceptable salt thereof. Thesynthesis of DMH1 as well as the structure and synthesis of relatedcompounds, e.g., derivatives, including those with similar biologicalactivity are known in the art, see, e.g. WO 2012/115120; WO 2013/016452;WO 2013/163228; WO 2013/166488; WO2014/138088; WO 2014/176606; WO2014/200115; WO2014/062138; US 2014/0248696; and U.S. Pat. No.8,822,684; each of which is incorporated by reference herein in itsentirety.

As used herein, “istodax” or “romidepsin” refers to a compound havingthe structure of Formula XIV or a pharmaceutically acceptable saltthereof. The synthesis of istodax as well as the structure and synthesisof related compounds, e.g., derivatives, including those with similarbiological activity are known in the art, see, e.g., WO14/102731;WO12/009336; WO13/106696; WO02/20817; U.S. Pat. Nos. 4,977,138;7,611,721; 7,608,280; and US 2012/046442; and J. Am. Chem. Soc. 118:7237-7238, 1996; each of which is incorporated by reference herein inits entirety.

Other terms are defined herein within the description of the variousaspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that HSC potential is rapidly lost upon culture. Peripheralbleed analysis following transplantation into lethally irradiated hostsof freshly isolated murine HSCs or HSCs cultured for 12 days ex vivo inS-clone+IL12/SCF/TPO+0.75% BSA. Note that the cultured cells do notretain the ability to give rise to peripheral blood donor chimerism thatis multi-lineage (B-cells, T-cells, myeloid cells, granulocytes) whereasfreshly transplanted HSCs give rise to robust levels of donor peripheralblood chimerism comprised of all of the blood lineages analyzedincluding B-cells, T-cells, myeloid cells, granulocytes.

FIG. 2 shows that engineered Fgd5-ZsGreen reporter mouse faithfullylabels HSCs. Bone marrow cells from Fgd5-ZsGreen reporter mouse wasenriched for cKit+ cells using magnetic beads and analyzed by flowcytometry for ZsGreen expression and a panel of markers (Lineage(Ter119, CD3, CD4, CD8, B220, Mac1, Gr1, Il7RA), cKit, Sca1, CD150,CD48). ZsGreen expression is shown for the indicated stem/progenitorsubpopulations. More details on the construction, characterization andspecificity of the Fgd5-ZsGreen HSC reporter mouse can be found in;Gazit R, Mandal P K, Ebina W, Ben-Zvi A, Nombela-Arrieta C, SilbersteinL E, Rossi D J. Journal of Experimental Medicine, 211(7):1315-31 (2014).

FIGS. 3A-3B show that Fgd5·ZsGr+ marks HSC potential during ex vivoculturing of HSCs. FIG. 3A depicts an overview: Fgd5·ZsGr+ HSCs wereisolated from reporter mice and cultured in S-clone+IL12/SCF/TPO+0.75%BSA for 4 days. On day four, 300 ZsGr+ and 300 ZsGr− cells were sortedand competitively transplanted into lethally irradiated mice showingthat all HSC activity was retained in the ZsGr+ fraction as detailed inFIG. 3B. FIG. 3B depicts peripheral blood chimerism and peripheral bloodgranulocyte chimerism of mice transplanted with ZsGr+ and ZsGr− cells.Lineage contribution of ZsGr+ and ZsGr− cells. Data shows that onlyZsGr+ cells retain HSC activity.

FIG. 4 shows a schematic representation of a small molecule screen forcompounds that support HSC ex vivo maintenance and expansion. 1)Fgd5·ZsGr+ HSC reporter mouse marrow was used to isolate ZsGr+ HSCs. 2)Isolation of HSCs marked by ZsGr reporter. 3) Development of assay wherehit is defined by the maintenance of HSC reporter expression in thecultured HSCs above DMSO control following 6 days ex vivo culture. 4)Hit compounds were then functionally validated in in vitro assays and invivo transplantation assays.

FIGS. 5A-5B show the development of a sensitivity assay. FIG. 5Ademonstrates that a total of 200 ZsGr+ HSCs (derived from theFgd5·ZsGreen HSC reporter mouse) and ZsGr−HSCs (derived from wild typemice not bearing the Fgd5Zs+ reporter) were seeded/well in variousratios (1:0; 1:1; 1:10; 1:20, 1:100, 0:1—shown as percentage ZsGr+) andimaged using the Operetta (Perkin Elmer) following 2 days ex vivoculture with individual cells plotted as being above or below thethreshold of ZsGreen detection. FIG. 5B demonstrates that after 2 daysof culture, the percentage of ZsGreen+ cells was determined. Thisestablished the analysis parameters and sensitivity for robust detectionof ZsGr+ signal after 2 days of culturing.

FIG. 6 shows a breakdown of pathways targeted in the primary smallmolecule screens.

FIGS. 7A-7B show initial screen results for various small molecule andgrowth factor libraries. FIG. 7A demonstrates the number of compoundsscreened, initial hits (that showed dose response), and validated hits(by flow cytometry to quantify ZSGr+) from each of 4 different librariesof small molecules targeting kinases, epigenetic regulators, andG-protein coupled receptors (GPCR), as well as a peptide library ofgrowth factors. FIG. 7B depicts representative results from 6-point doseresponse (10 uM, 5 uM, 1 uM, 0.5 uM, 0.1 uM, 0.05 uM). * indicates ahit.

FIG. 8 shows the ex vivo culture of 20 murine HSCs(Lineage−Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+) for 7 or 14 days in thepresence of DMSO, LSD1 inhibitor IV (LS), Tgfbeta inhibitor (RepSox) orthe combination of both (C2). Images taken at 4× magnification. Notethat the cultures in the presence of LS and C2 are more homogenous andless differentiated.

FIGS. 9A-9B show the expansion of phenotypic murine HSCs for 14 days exvivo. 20 murine HSCs (Lineage−Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+) werecultured in the presence of DMSO, LSD1 inhibitor IV (LS), Tgfbetainhibitor RepSox (RS), and the combination of both (C2) for 14 days.This method supports maintenance and expansion of ZsGr+ HSCs. FIG. 9Adepicts representative FACS plots of HSCs cultured for 14 days ex vivoin the presence of DMSO and the combination of LSD1 inhibitor (LS),Tgfbeta inhibitor RepSox (RS) (C2) showing increased levels ofphenotypic HSCs in the presence of C2. FIG. 9B depicts the number ofLineage−Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+ HSCs in each conditionafter 14 days of ex vivo culture.

FIGS. 10A-10C show that LSD1 inhibitor (LS), Tgfbeta inhibitor RepSox(RS), and the combination of both (C2) supports maintenance andexpansion of ZsGr+ murine HSCs. FIG. 10A depicts bright-field and ZsGrimages of 20 Fgd5·ZsGr+ HSCs cultured for 4.5 days ex vivo in thepresence of LSD1 inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS), andthe combination of both (C2). FIG. 10B demonstrates the frequency ofZsGr+ cells remaining following 4.5 days of ex vivo culture. FIG. 10Cdepicts the number of ZsGr+ and ZsGr− cells after 4.5 day cultures.

FIGS. 11A-11B show the results of a colony forming assay of purifiedmurine HSCs exposed to DMSO, LSD1 inhibitor (LS), Tgfbeta inhibitorRepSox (RS), and the combination of both (C2) cultured for 14 days. HSCswere cultured in the presence of DMSO, LSD1 inhibitor IV (LS), Tgfbetainhibitor RepSox (RS), and the combination of both (C2) for 14 days andthen 1/300 of the culture was plated in 1 ml of MethoCult 3434. Arisingcolonies were scored 10 days post-plating into methylcellulose. FIG. 11Adepicts colony number and composition. FIG. 11B depicts the total numberof cells generated by HSCs cultured for 14 days.

FIGS. 12A-12D show the results of competitive transplantation ofpurified murine HSCs (lineage-Sca1+ckit+CD150+CD48−CD34−Fgd5ZsGr+)exposed to DMSO, LSD1 inhibitor (LS), Tgfbeta inhibitor RepSox (RS), andthe combination of both (C2) cultured for 14 days. The cultures arisingfrom 100 starting murine HSC were competitively transplanted intolethally irradiated recipients following 14 days of ex vivo cultureDMSO, LSD1 inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS), and thecombination of both (C2) showing FIG. 12A depicts a graph of peripheralblood donor chimerism, and FIG. 12B depicts a graph of peripheral blooddonor (CD45.2) granulocyte chimerism. FIG. 12C depicts a graph ofindividual recipient mouse donor chimerism, and FIG. 12D depicts a graphof lineage contribution of donor reconstitution showing B-cell (B220+),T-cell (CD3+) and myeloid cell (Mac1+) at week 24 post-transplant isshown.

FIGS. 13A-13F show the results of a limit dilution assay of purifiedmurine HSCs (lineage−Sca1+ckit+CD150+CD48−CD34−Fgd5ZsGr+) cultured for14 days with the combination (C2) of LSD1 inhibitor IV (LS) and theTgfbeta inhibitor RepSox (RS). The cultures arising from 1, 5, 20, 50 or200 starting murine HSCs were competitively transplanted following 14days of ex vivo culture in the presence of C2. FIG. 13A depictsperipheral blood donor chimerism. FIG. 13B depicts peripheral blooddonor (CD45.2) granulocyte Chimerism. FIG. 13C depicts the lineagecontribution of donor reconstitution showing B-cell (B220+), T-cell(CD3+) and myeloid cell (Mac1+), FIG. 13D depicts donor chimerism ofindividual recipients, and FIG. 13E depicts granulocyte chimerism ofindividual recipients at 20 weeks post-transplant. FIG. 13F depictscalculation of limit dilution frequencybioinf.wehi.edu.au/software/elda/Hu, Y, and Smyth, G K (2009). ELDA:Extreme limiting dilution analysis for comparing depleted and enrichedpopulations in stem cell and other assays. Journal of ImmunologicalMethods 347, 70-78.

FIGS. 14A-14B show secondary small molecule screen for compounds thatsynergize with C2 (LSD1 inhibitor IV and Tgfbeta inhibitor) to supportHSC ex vivo maintenance and expansion. Schematic of primary (FIG. 14A)and (FIG. 14B) secondary screen in which 124 potential hit compoundsidentified in primary screen were rescreened (screen 2) in the presenceof C2.

FIGS. 15A-15C show potential hits of secondary screen that targetpathways of interest including Tgfbeta, histone methylation, histoneacetylation, p38 signaling and Wnt signaling. FIG. 15A depicts anoverview of two strategies used to identify hits in secondary smallmolecule screen (C2 is combination of LSD1 inhibitor IV and the Tgfbetainhibitor RepSox). FIG. 15B depicts hits found by following a strategybased on ZsGreen+ HSC percentage (strategy 1). FIG. 15C depicts hitsfound by following a strategy based on number of ZsGreen+ HSCs (strategy2).

FIGS. 16A-16B show hits of a secondary screen of compounds identified ina primary screen and then re-screened in the presence of C2 (Combinationof LSD1 inhibitor IV and the Tgfbeta inhibitor RepSox). FIG. 16A depictshits found by following a strategy based on ZsGreen+ HSC percentage.FIG. 16B depicts hits found by following a strategy based on number ofZsGreen+ HSCs.

FIG. 17 shows the results of experiments testing compounds previouslyreported to maintain murine HSCs. Culturing ZsGreen positive HSCs for 6days in the presence of: dmPGE2 (North, Zon, Nature. 2007), BIO (Ko etal, Stem Cells. 2011), p38 inhibitor (Wang et al, Stem Cells Dev. 2011),DMSO: negative control. D2 is ZsGr+ HSCs maintained for 2 days ex vivo.Threshold of cells identified as ZsGreen+(i.e., HSCs) is shown witharrow.

FIGS. 18A-18B show a hypothesis driven strategy for modulating candidatepathways towards HSC ex vivo maintenance and expansion. FIG. 18A depictsselection of candidate target pathways via comparison of intestinal stemcell and hematopoietic stem cell maintenance and proliferation signals.FIG. 18B depicts selection of agents/pathway modulators.

FIG. 19 shows a schematic for assessing the activity of pathwaymodulators on HSC maintenance and expansion.Fgd5-ZsGreen+immunophenotypic HSCs(Lineage−cKit+Sca1+CD150+CD48-Fgd5·ZsGreen+) were sorted and cultured inthe presence of cytokines only (Standard media) or additionallysupplemented with 7 candidate pathway modulators (W7 media). Compoundsmodulating the 7 pathways are: A83-01, Tranylcypromine, Trichostatin A,SB203580, CHIR99021, DMH1, Sodium acetate (called here Supplements).Multiparametric analysis of cellular immunophenotype was performed byflow cytometry after 14 days of culture.

FIG. 20 shows the combinatorial modulation of 7 candidate pathwaysmaintains and expands immunophenotypic HSCs during ex vivo culturing. 50mouse HSCs were cultured in a serum free media supplemented with SCF,TPO, and IL12, plus or minus the seven candidate pathway modulators.Flow cytometric analysis was performed on Day 15. Compounds modulatingthe 7 pathways are: A83-01, Tranylcypromine, Trichostatin A, SB203580,CHIR99021, DMH1, and Sodium acetate.

FIGS. 21A-21B show the contribution of each compound/pathway in abilityto maintain and expand phenotypic HSCs during ex vivo culture. 50 murineHSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured inthe presence of cytokines only (SCF, TPO, and IL12) in the absence ofcompounds (standard), or with all 7 compounds (W7), or with subtractionof each individual compound (A83-01 (A), Tranylcypromine (TC),Trichostatin A (TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH),Sodium acetate (OAC)). Flow cytometry analysis was performed on Day 14showing (FIG. 21A) differentiation to lineage+ cells (stained byantibody cocktail against antigens for: B-cells, T-cells, myeloid cells,erythrocytes, and granulocytes), where lineage positive is to the rightof the dashed line. FIG. 21B depicts absolute HSC numbers after 14 daysculture from 50 starting HSCs in the indicated conditions.

FIGS. 22A-22B show that structurally distinct HDAC inhibitors functionequivalently to maintain immunophenotypic HSCs. 100 murine HSCs(lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured in thepresence of cytokines only (SCF, TPO, and IL12) in the absence ofcompounds (Standard), or additionally supplemented with a cocktail ofcompounds (Lithium chloride, nicotinamide, N-acetylcysteine, ascorbicacid, A83-01, and SB203580) plus either valproic acid (VPA) ortrichostatin A (TSA), which are structurally distinct HDAC inhibitors.FIG. 22A depicts day 7 flow cytometric analysis and FIG. 22B depicts theproportion of Fgd5-ZsGreen+Sca1+ cells for each replicate.

FIG. 23 shows that the supplementation with additional compounds duringex vivo culturing reduces heterogeneity of Fgd5·ZsGr+ HSCs cells withrespect to CD48 and Sca1 expression. 40 murine HSCs (lineage-, ckit+,Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 12 days in thepresence of cytokines (SCF, TPO, and IL12) and a cocktail of compounds(Lithium chloride, nicotinamide, N-acetylcysteine, ascorbic acid,A83-01, and SB203580, trichostatin A) plus either or both DNAmethyltransferase inhibitor (RG108) and G9a inhibitor (UNC0638). Flowcytometry plots of Fgd5+Lineage− cells from the indicated cultureconditions are shown. The histogram shows the proportions of theindicated subpopulations.

FIG. 24 shows the in vivo function of murine HSCs cultured for 14 daysin the presence of DMSO (Standard) or compounds targeting 7 pathways(Combination) consisting of Tgfbeta inhibitor A83-01, Lsd1 inhibitorTranylcypromine, HDAC inhibitor Trichostatin A, the p38 kinase inhibitorSB203580, BMP inhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodiumacetate. 10 HSCs were cultured for 14 days in the indicated conditionsfollowed by in vivo competitive transplantation into lethally irradiatedhosts (against 2×10⁵ congenically marked bone marrow cells). Peripheralblood donor chimerism at indicated time points post-transplantation areshown.

FIGS. 25A-25C show the in vivo function of murine HSCs cultured for 14days in the presence of DMSO (S: Standard), or compounds targeting 7pathways (W7: Tgfbeta inhibitor A83-01, Lsd1 inhibitor Tranylcypromine,HDAC inhibitor Trichostatin A, the p38 kinase inhibitor SB203580, BMPinhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodium acetate). 100HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were culturedfor 14 days in the indicated conditions followed by in vivo competitivetransplantation (against 2×10⁵ congenically marked bone marrow cells).FIG. 25A depicts peripheral blood and FIG. 25B depicts granulocyte donorchimerism at indicated time points post-transplantation are shown. FIG.25C depicts donor HSC chimerism in the bone marrow of transplantrecipients transplanted with HSCs cultured for 14 days in the indicatedconditions is shown.

FIG. 26 shows that the modulation of four pathways is sufficient tomaintain/expand immunophenotypic murine HSCs. 50 HSCs (lineage-, ckit+,Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 14 days in serum freemedia supplemented with cytokines in the presence of DMSO, or compoundstargeting 4 pathways (W4: Tgfbeta inhibitor A83-01, Lsd1 inhibitorTranylcypromine, HDAC inhibitor Trichostatin A, and the p38 kinaseinhibitor SB203580) identified from the initial set of 7 compounds(Tgfbeta inhibitor A83-01, Lsd1 inhibitor Tranylcypromine, HDACinhibitor Trichostatin A, the p38 kinase inhibitor SB203580, BMPinhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodium acetate)Immunophenotypic HSCs (Lineage−cKit+Sca1+CD48−CD150+Fgd5ZsGreen+CD41-)were analyzed by flow cytometry.

FIG. 27 shows a sorting strategy for primary human HSCs from CD34+enriched cord blood. Sorted HSCs have the immunophenotype ofCD34+lineage-CD38-CD45RA-CD90+.

FIGS. 28A-28B show that the combination (C2) of LSD1 inhibitor IV (LS)and the Tgfbeta inhibitor RepSox (RS) can maintain and expand phenotypichuman cord blood (CB) HSCs following 14 days ex vivo culturing. FIG. 28Adepicts a schematic of experimental design. FIG. 28B depicts flowcytometry of cultured cord blood HSCs (CD34+CD45RA-CD90+CD38-, initiallyseeded at 50 cells per well) 14 days post-plating in the presence ofDMSO, Stem Regenin 1 (SR1), LSD1 inhibitor (LS), Tgfbeta inhibitorRepSox (RS) and the combination of LSD1 inhibitor IV and RepSox (C2)showing percentages of CD34+CD45RA− cells (contour plots) and CD90+cells (histogram) (+/− standard deviation).

FIGS. 29A-29B show that the combination (C2) of LSD1 inhibitor IV (LS)and the Tgfbeta inhibitor RepSox (RS) can maintain and expand phenotypichuman cord blood HSCs following 14 days ex vivo culturing. FIG. 29Adepicts the percentage of HSCs (as defined as CD34+CD45RA-CD90+) oftotal live cells and FIG. 29B depicts absolute numbers of HSCs, postculturing 50 cord blood HSCs (CD34+CD45RA-CD90+CD38-) in the presence ofDMSO, Stem Regenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfbeta inhibitorRepSox (RS) and the combination of LSD1 IV inhibitor and RepSox (C2).*p<0.05 unpaired t-test.

FIGS. 30A-30B show that the combination (C2) of LSD1 inhibitor IV (LS)and the Tgfbeta inhibitor RepSox (RS) maintains and expands primitive invitro colony forming potential of human cord blood HSCs following 14days of ex vivo culture. FIG. 30A depicts colony counts and compositionadjusted to represent the entire well following 14 days of ex vivoculture (Note: only a fraction of the well was added to methocult forcolony formation). FIG. 30B depicts the frequency of myeloid colonytypes from 14 day cord blood HSC cultures. Note elevated frequency ofmost primitive GEMM colonies (colonies comprised of granulocytes,macrophages, erythroid and megakaryocytic lineages) after culturing inRS, LS or C2.

FIGS. 31A-31B show that the combination (C2) of LSD1 inhibitor IV (LS)and the Tgfbeta inhibitor RepSox (RS) can maintain and expand phenotypichuman bone marrow HSCs following 14 days ex vivo culturing. FIG. 31Adepicts flow cytometry of cultured bone marrow derived HSCs (FACSpurified as CD34+CD45RA-CD90+CD38-, 80 cells per well) 14 dayspost-plating in the presence of DMSO, Stem Regenin 1 (SR1), LSD1inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS) and the combination(C2) of LSD1 inhibitor IV and RepSox showing percentages of CD34+CD45RA−cells (dot plots) and CD90+ cells (histogram) (+/− Standard deviation).FIG. 31B depicts the percentage of immunophenotypic HSCs(CD34+CD45RA-CD90+CD38-) post culturing in the presence of DMSO, StemRegenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS)and the combination (C2) of LSD1 inhibitor IV and RepSox. *p<0.05, **p<0.005 unpaired t-test.

FIGS. 32A-32B show that the combination (C2) of LSD1 inhibitor IV (LS)and the Tgfbeta inhibitor RepSox (RS) can maintain and expand phenotypichuman mobilized peripheral blood HSCs following 14 days ex vivoculturing. FIG. 32A depicts flow cytometry of cultured mobilizedperipheral blood HSCs (FACS purified as CD34+CD45RA-CD90+CD38-, 50 cellsper well) 14 days post-plating in the presence of DMSO, Stem Regenin 1(SR1), LSD1 inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS) and thecombination (C2) of LSD1 inhibitor IV and RepSox showing percentages ofCD34+CD45RA-cells (contour plots) and CD90+ cells (histogram) (+/−Standard deviation). FIG. 32B depicts the percentage of immunophenotypicHSCs (CD34+CD45RA-CD90+) and stem and progenitors (CD34+CD45RA−) post 14days culture in the presence of DMSO, Stem Regenin 1 (SR1), LSD1inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS) and the combination(C2) of LSD1 inhibitor IV and RepSox.

FIGS. 33A-33B show that compounds targeting 7 pathways identified inmurine system enable maintenance and expansion of immunophenotypic cordblood HSCs. 200 cord blood HSCs (FACS purified asLineage-CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum freemedia supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presenceof DMSO, or compounds targeting 7 pathways (Combination: Tgfbetainhibitor A83-01, Lsd1 inhibitor Tranylcypromine, HDAC inhibitorTrichostatin A, the p38 kinase inhibitor SB203580, BMP inhibitor DMH1,Gsk3beta inhibitor Chir99021, and sodium acetate) showing (FIG. 33A)Immunophenotype of the cells post-culturing analyzed by flow cytometryand (FIG. 33B) quantification of immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38−CD90+).

FIGS. 34A-34B show that targeting 3 pathways is sufficient formaintenance and expansion of immunophenotypic human cord blood HSCs. 200cord blood HSCs (FACS purified as Lineage-CD34+CD45RA-CD90+CD38-) werecultured for 12 days in serum free media supplemented with cytokines(SCF, TPO, FLT3L, IL3) in the presence of the indicated chemicalcombinations (Tgfbeta inhibitor (A, A83-01), HDAC inhibitor (TSA,Trichostatin A), and LSD1 inhibitor (TC, Tranylcypromine)) and analyzedby flow cytometry. FIG. 34A depicts the immunophenotype of the cellspost-culturing, and FIG. 34B depicts quantification of immunophenotypicHSCs (Lineage−CD34+CD45RA-CD38−CD90+) cultured in the indicatedconditions.

FIGS. 35A-35B show that supplementation of minimal chemical combinationwith p38 inhibitor improves the yield of human cord blood HSCs. 200 cordblood HSCs (FACS purified as Lineage-CD34+CD45RA-CD90+CD38-) werecultured for 12 days in serum free media supplemented with cytokines(SCF, TPO, FLT3L, IL3) in the presence of the indicated chemicalcombinations (Tgfbeta inhibitor (A, A83-01), HDAC inhibitor (TSA,Trichostatin A), and LSD1 inhibitor (TC, Tranylcypromine)) oradditionally supplemented with p38 inhibitor (p38i, SB203580) andanalyzed by flow cytometry. FIG. 35A depicts the immunophenotype of thecells post-culturing, and FIG. 35B depicts quantification ofimmunophenotypic HSCs (Lineage−CD34+CD45RA-CD38−CD90+CD49F+) cultured inthe indicated conditions.

FIG. 36 shows the cultivation under low oxygen tension improves theyield of human cord blood HSCs. 200 cord blood HSCs (FACS purified asLineage-CD34+CD45RA-CD90+CD38-) were cultured in serum free mediasupplemented with cytokines (SCF, TPO, FLT3L, IL3) and compoundstargeting 3 pathways (W3: Tgfbeta inhibitor (A83-01), HDAC inhibitor(Trichostatin A), and LSD1 inhibitor (Tranylcypromine)) for 12 days ineither standard tissue culture incubator (atmospheric oxygen, 21% O2) orlow oxygen incubator (5% O2). Immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38−CD90+) cultured in the indicated conditionswere quantified post-culturing.

FIGS. 37A-37C show a comparison of chemical combination with compoundspreviously reported to expand cord blood HSCs. 200 cord blood HSCs (FACSpurified as Lineage-CD34+CD45RA−CD90+CD38-) cultured for 12 days inserum free media supplemented with cytokines (SCF, TPO, FLT3L, IL3) inthe presence of StemRegenin1 (SR1), UM171, or chemical combination (W3:Tgfbeta inhibitor (A83-01), HDAC inhibitor (Trichostatin A), and LSD1inhibitor (Tranylcypromine)) and analyzed by flow cytometry. FIG. 37Adepicts the immunophenotype of the cells post-culturing, FIG. 37Bdepicts the percentage of indicated populations, and FIG. 37C depictsquantification of immunophenotypic HSCs (Lineage−CD34+CD45RA-CD38−CD90+)12 days post-culturing in the indicated conditions.

FIGS. 38A-38C show the ex vivo maintenance and expansion of humanmobilized peripheral blood CD34+ cells using compounds identified usingmurine cells. 3000 CD34+ enriched mobilized peripheral blood cells werecultured for 7 days in serum free media supplemented with cytokines(SCF, TPO, FLT3L, IL3) in the presence of the indicated individualchemicals or chemical combinations (Tgfbeta inhibitor (A, A83-01), HDACinhibitor (TSA, Trichostatin A), LSD1 inhibitor (TC, Tranylcypromine),and p38 inhibitor (p38i, SB203580)) and analyzed by flow cytometry. FIG.38A depicts the immunophenotype of the cells post-culturing, FIG. 38Bdepicts the percentage of indicated populations, and FIG. 38C depictsthe quantification of immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38−CD90+CD49F+) post-culturing in the indicatedconditions for 7 days. (W7: A83-01 (A), Tranylcypromine, (TC)Trichostatin A (TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH),Sodium acetate (OAC), and W3: A83-01 (A), Tranylcypromine, (TC)Trichostatin A (TSA))

FIG. 39 shows that the ex vivo culture of human mobilized peripheralblood CD34+ cells using chemical combination enriches immunophenotypicHSCs. 3000 CD34+ enriched mobilized peripheral blood cells were culturedfor 7 days in serum free media supplemented with cytokines (SCF, TPO,FLT3L, IL3) in the presence of DMSO, the indicated individual chemicals(StemRegenin (SR1), UM171) or combination of four compounds (W4: Tgfbetainhibitor (A83-01), HDAC inhibitor (Trichostatin A), LSD1 inhibitor(Tranylcypromine), and p38 inhibitor (SB203580)) and analyzed by flowcytometry. Quantification of the fraction of immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38−CD90+) in CD34+ enriched mobilized peripheralblood prior to ex vivo culture (Uncultured) and post-culturing in theindicated conditions for 7 days showing a 8-fold enrichment of HSCspost-culturing.

FIGS. 40A-40C show a comparison of chemical combination with compoundspreviously reported to expand cord blood HSCs for their ability tomaintain/expand human mobilized peripheral blood HSCs. 50 mobilizedperipheral blood HSCs (FACS purified as Lineage-CD34+CD45RA−CD90+CD38-)were cultured for 12 days in serum free media supplemented withcytokines (SCF, TPO, FLT3L, IL3) in the presence of StemRegenin1 (SR1),UM171, or chemical combination (W3: Tgfbeta inhibitor (A83-01), HDACinhibitor (Trichostatin A), and LSD1 inhibitor (Tranylcypromine)) oradditionally supplemented with p38 inhibitor (p38i, SB203580) andanalyzed by flow cytometry. FIG. 40 A depicts the immunophenotype of thecells post-culturing, FIG. 40B depicts the percentage of indicatedpopulations, and FIG. 40C depicts quantification of immunophenotypicHSCs (Lineage−CD34+CD45RA-CD38−CD90+CD49F+) post culturing in theindicated conditions for 12 days.

FIGS. 41A-41C show that Romidepsin, a HDAC1/2 specific inhibitor, canreplace the pan HDAC inhibitor Trichostatin A for efficient ex vivomaintenance/expansion of human HSCs. 3000 CD34+ enriched mobilizedperipheral blood cells were cultured for 7 days in serum free mediasupplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence ofthe indicated chemical combinations (Tgfbeta inhibitor (A, A83-01),pan-HDAC inhibitor (TSA, Trichostatin A), LSD1 inhibitor (TC,Tranylcypromine), p38 inhibitor (p38i, SB203580), HDAC1/2 inhibitor(Rom, Romidepsin)) and analyzed by flow cytometry. FIG. 41A depicts theimmunophenotype of the cells, FIG. 41B depicts the percentage ofindicated populations, and FIG. 41C depicts quantification ofimmunophenotypic HSCs (Lineage−CD34+CD45RA-CD38−CD90+CD49F+) 7 dayspost-culturing in the indicated conditions.

FIGS. 42A-42C show transplantation results of human CD34+ cord bloodcells cultured for 14 days ex vivo. The cultures of 10,000 startingCD34+ cord blood cells were transplanted into sublethally irradiatedimmunocompromised NSG (Nod-Scid-gamma) mice following 14 days of ex vivoculture in the presence of DMSO, W3 (Tgfbeta inhibitor A83-01, LSD1inhibitor tranylcypromine, HDAC inhibitor trichostatin A), thecombination of LSD1 inhibitor IV and the Tgfbeta inhibitor RepSox (C2),Stem Regenin 1 (SR1) and UM171; or 10,000 uncultured CD34+ cord bloodcells (fresh) showing (FIG. 42A) peripheral blood donor chimerism. FIG.42B depicts quantification of peripheral blood donor chimerism at weeks24 and 30 post transplant. FIG. 42C depicts lineage contribution oftransplanted cells at week 30 post-transplant.

FIG. 43 depicts a schematic of the experimental procedure for theresults depicted in FIGS. 44-48C.

FIG. 44 depicts the results of FACS analysis 12 days post culturing.

FIG. 45 depicts a graph of total live cell output 12 days post-culturingfrom 300 starting cell HSC equivalents in the presence of DMSO, W7, orW3.

FIGS. 46A-46B depict graphs of the frequency (FIG. 46A) and absolutenumber (FIG. 46B) of lineage-IL7R-ckit+Sca1+(LSK) cells 12 dayspost-culturing in the presence of DMSO, W7, or W3.

FIGS. 47A-47B depicts graphs of the frequency (FIG. 47A) and absolutenumber (FIG. 47B) of lineage-IL7R-ckit+Sca1+CD48-CD150+ HSCs 12 dayspost-culturing in the presence of DMSO, W7, or W3.

FIGS. 48A-48C depict an experiment schematic (FIG. 48A), a graph ofdonor cell engraftment (FIG. 48B) and a graph of lineage contribution(FIG. 48C) 4 weeks post-transplantation of 200 starting cell (HSC)equivalents cultured for 12 days in the presence of DMSO, W7, or W3.Lineage analysis of Mac1+ myeloid cells, CD3 positive (3) T-cells andB220 positive B-cells.

DETAILED DESCRIPTION

The present invention is based on the surprising discovery that the exvivo expansion, enrichment, and maintenance of populations ofhematopoietic stem cells bearing hematopoietic stem cell functionalpotential can be achieved by contacting these cells with one or moreagents that exhibit one, two, or more activities selected from the groupconsisting of modulation of histone methylation, inhibition of TGFβsignaling, inhibition of p38 signaling, activation of canonical Wntsignaling, and modulation of histone acetylation. A wide variety ofstructurally and mechanistically distinct agents that modulate thesebiological events are known in the art. For instance, these agents maybe small molecules capable of agonizing or antagonizing a particularevent within a certain pathway (e.g., small molecules that inhibitenzymatic activity of proteins that propagate a signal transductioncascade). These agents may also be antibodies, such as monoclonalantibodies or antigen-binding fragments thereof, that disrupt aparticular interaction (e.g., a ligand-receptor interaction) by virtueof competitively binding a particular protein and sterically precludingthe association of the protein with its cognate binding partner. Otheragents, such as therapeutic proteins and structurally constrainedpeptides, are topologically well-suited for antagonizing protein-proteininteractions that occur over larger molecular surfaces and thusrepresent a class of inhibitors capable of intervening within signaltransduction pathways at targets that have otherwise been intractable todisrupting with conventional small molecule therapeutics. Other classesof inhibitors include interfering RNA molecules, which are capable ofattenuating the expression of a target gene by binding mRNApolynucleotides via complementary hydrogen bonding and, e.g., inducingthe degradation of the target mRNA or sterically preventing thenucleation of ribosome assembly. The sections that follow provide anoverview of examples of the types of agents useful with the compositionsand methods of the invention so as to promote hematopoietic stem cellexpansion, enrichment, and maintenance of hematopoietic stem cellfunctional potential.

UM171 and Structural Analogs Thereof

Additional agents that can be used in conjunction with the methods ofthe invention include UM171, a small molecule that has been shown toinduce hematopoietic stem cell expansion. UM171 is described, e.g., inFares et al. Science 345:1509 (2014), the disclosure of which isincorporated herein by reference. Other agents that can be used toexpand, enrich, and maintain hematopoietic stem cells include UM171analogs, such as a UM171 structural analogs according to any one ofFormulas (I), (II), (III), (IV), (V), and (VI) of US 2015/0011543, thedisclosure of which is incorporated herein by reference. For instance,analogs of UM171 that can be used in conjunction with the compositionsand methods described herein include compounds listed in Table 11,below.

TABLE 11 UM171 and structural analogs thereof Com- pound No. Molecularstructure 100 (UM171)

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

Small Molecules

A variety of small molecules may be used in conjunction with the methodsdescribed herein. Among these include modulators of enzyme-substrateinteractions. A variety of small molecules have been developed in orderto antagonize enzyme-substrate interactions or to intervene at distinctpoints within a signal transduction cascade. For instance,tranylcypromine and derivatives thereof represent a robust class ofinhibitors capable of irreversibly binding to and inhibiting histonedemethylases such as LSD1 by virtue of forming a covalent adduct withthe isoalloxazine moiety of the FAD cofactor utilized by these enzymesto catalyze oxidative demethylation of N-methylated histone tailresidues. Exemplary small molecule inhibitors of histone demethylationuseful with the compositions and methods of the invention include LSD1inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76,LSD1 inhibitor III CBB1007, LSD1 inhibitor I (also referred to as BHC110Inhibitor I, Histone Lysine Demethylase Inhibitor III, and/or KDM1Inhibitor I), and tranylcypromine, described above. Examples of smallmolecules useful for inhibiting histone demethylases additionallyinclude phenelzine, propargylamine, and derivatives thereof as describedin US 2013/0095067, the disclosure of which is incorporated herein byreference. Other tranylcypromine derivatives have been described, e.g.,in US 2014/0163041, the disclosure of which is incorporated herein byreference.

Additional examples of small molecules that can be used to modulatehistone methylation include BIX01294 (a H3K9 methylation inhibitordescribed in, e.g., WO 2014/057997); UNC0638 (a H3K9 methylationinhibitor described in, e.g., WO 2013/050422), the disclosures of eachof which are incorporated herein by reference; and CARM1 Inhibitor (PRMTInhibitor V,3,5-bis(3-Bromo-4-hydroxybenzylidene)-1-benzylpiperidin-4-one, a histonearginine methyltransferase inhibitor).

Several structurally distinct classes of small molecules inhibitors ofTGFβ signaling have been reported. These agents can be classified on thebasis of the core molecular scaffolds of these molecules. For example,TGFβ signaling inhibitors may contain a dihydropyrrlipyrazole,imidazole, pyrazolopyridine, pyrazole, imidazopyridine, triazole,pyridopyrimidine, pyrrolopyrazole, isothiazole or oxazole functionalityas the core structural fragment of the molecule. Some non-limitingexamples of small molecule inhibitors of TGFβ signaling include ALK5inhibitor II (also referred to as E-616452), LY364947 (also referred toas ALK5 Inhibitor I, TbR-I Inhibitor, Transforming Growth Factor-b TypeI Receptor Kinase Inhibitor), A83-01, and DMH1, described above. Otherexamples of small molecules that can be used to modulate TGFβ signalingin conjunction with the compositions and methods of the inventioninclude SB431542(4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamidehydrate,4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamidehydrate,4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamidehydrate, an Alk5 inhibitor), Galunisertib (LY2157299, an Alk5inhibitor), LY2109761(4-[2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine,an Alk5/TGFβRII inhibitor), SB525334(6-[2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl]quinoxaline,an Alk5 inhibitor), GW788388(N-(oxan-4-yl)-4-[4-(5-pyridin-2-yl-1H-pyrazol-4-yl)pyridin-2-yl]benzamide,an Alk5 inhibitor), K02288(3-[6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl]phenol, an Alk4/Alk5inhibitor), SD-208(2-(5-chloro-2-fluorophenyl)-N-pyridin-4-ylpteridin-4-amine, an Alk5inhibitor), EW-7197(N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline,an Alk4/Alk5 inhibitor), andLDN-212854(5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline,an Alk4/Alk5 inhibitor).

Additional examples of small molecule TGFβ modulators includeantagonists of TGFβ receptors, such as2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole.Other small molecule inhibitors include, but are not limited to,SB-431542,(4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide,described in Halder et al. Neoplasia 7:509 (2005)), SM16, a smallmolecule inhibitor of TGFβ receptor ALK5, the structure of which isshown below (Fu et al. Arteriosclerosis, Thrombosis and Vascular Biology28:665 (2008)), SB-505124 (an Alk4/Alk5 inhibitor, structure shownbelow, described in Dacosta Byfield et al. Molecular Pharmacology 65:744(2004)), and 6-bromo-indirubin-3′-oxime (described in U.S. Pat. No.8,298,825), the disclosures of each of which are incorporated herein byreference.

Additional examples of inhibitors of TGF-β signaling are described in,e.g., Callahan et al. Journal of Medicinal Chemistry 45:999 (2002);Sawyer et al. Journal of Medicinal Chemistry 46:3953 (2003); Gellibertet al. Journal of Medicinal Chemistry 47:4494 (2004); Tojo et al. CancerScience 96:791 (2005); Petersen et al. Kidney International 73:705(2008); Yingling et al. Nature Reviews Drug Discovery 3:1011 (2004);Byfield et al. Molecular Pharmacology 65:744 (2004); Dumont et al.Cancer Cell 3:531 (2003); WO 2002/094833; WO 2004/026865; WO2004/067530; WO 2009/032667; WO 2004/013135; WO 2003/097639; WO2007/048857; WO 2007/018818; WO 2006/018967; WO 2005/039570; WO2000/031135; WO 1999/058128; U.S. Pat. Nos. 6,509,318; 6,090,383;6,419,928; 7,223,766; 6,476,031; 6,419,928; 7,030,125; 6,943,191; US2005/0245520; US 2004/0147574; US 2007/0066632; US 2003/0028905; US2005/0032835; US 2008/0108656; US 2004/015781; US 2004/0204431; US2006/0003929; US 2007/0155722; US 2004/0138188; and US 2009/0036382, thedisclosures of each which are incorporated herein by reference.

Another class of small molecules useful with the compositions andmethods of the invention include modulators of bone morphogeneticprotein (BMP) signaling. BMP is a member of the TGF superfamily ofligands, and modulators of BMP signaling, such as inhibitors of Alk2,Alk3, and Alk6, can be used in conjunction with the methods of theinvention, e.g., to expand, enrich, and/or maintain hematopoietic stemcells in a multi-potent state. Exemplary BMP inhibitors include DMH1(4-[6-(4-Isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline,4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline),K02288 (3-(6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol),LDN-212854(5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline),LDN-193189(4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline),LDN-214117(1-(4-(6-Methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine),and ML347(5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline).

Inhibitors of receptor tyrosine kinases, such as vascular endothelialgrowth factor (VEGF) and platelet-derived growth factor (PDGF) signalingcan also be used in conjunction with the compositions and methods of theinvention to promote hematopoietic stem cell expansion, enrichment, andmaintenance of hematopoietic stem cell functional potential. Forinstance, an exemplary VEGF/PDGF inhibitor useful with the methodsdescribed herein is ABT-869 (Linifanib,1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea).

Other small molecules useful with the compositions and methods of theinvention include inhibitors of DNA methylation, including chemicalmodulators of DNMT1, DNMT3a, and DNMT3B. An exemplary inhibitor of thesetargets that can be used in conjunction with the compositions andmethods of the invention to expand, enrich, and maintain thehematopoietic stem cell functional potential of hematopoietic stem cellsis RG108 (N-Phthalyl-L-tryptophan).

A variety of small molecule inhibitors of p38 MAPK have also beenreported to date, including the pyridinylimidazole compounds SB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)and SB202190 (4(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole). These compounds represent a class of inhibitors thatselectively antagonize p38 MAPK α- and β-isoforms without disrupting theenzymatic activity of the γ- or β-isoforms. These compounds aredescribed in U.S. Pat. No. 6,602,896, the disclosure of which isincorporated herein by reference. Other example of p38 MAPK inhibitorsinclude SB203580, BIRB796 (Doramapimod), VX702, SB202190, LY2228820,VX745, Vinorelbine (Navelbine), PH797804, pamapimod, CMPD-1, EO1428,JX401, ML3403, RWJ67657, SB239063, SCIO469 hydrochloride, SKF86002dihydrochloride, SX011, and TAK715, e.g., as described in US2014/0127231, the disclosure of which is incorporated herein byreference. Additional examples of p38 inhibitors useful with thecompositions and methods of the invention include Pexmetinib (ARRY-614),PH-797804(3-(3-Bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N,4-dimethylbenzamide),Losmapimod (GW856553X), and Skepinone-L.

Small molecule agents capable of inhibiting a protein that promotes thedegradation of β-catenin include those capable of attenuating theactivity of proteins that promote β-catenin phosphorylation. Suchinhibitors serve to increase the nuclear concentration of thistranscription factor, and a variety of examples are known in the art.Inhibitors of β-catenin phosphorylation include compounds that inhibitglycogen synthase kinase 3 (GSK3), such as CHIR99021, described above,as well as 6-bromo-indirubin-3′-oxime (Meijer et al. Chemistry andBiology 10:1255 (2003); Goessling et al. Cell 136:1136 (2009)),AR-A014418 (Bhat et al. Journal of Biological Chemistry 278:45937(2003), and the organometallic GSK-3 inhibitor DW21 (Williams et al.Angewandte Chemie International Edition 44:1984 (2005)), the disclosuresof which are incorporated herein by reference. Other small moleculemodulators of Wnt signaling useful in conjunction with the compositionsand methods of the invention to expand, enrich, and maintain thehematopoietic stem cell functional potential of hematopoietic stem cellsinclude inhibitors of GSK3a and GSK3b, such as CHIR99021 and Lithiumchloride.

Histone deacetylation is also amenable to targeting with small moleculetherapeutics. Hydroxamic acids represent a particularly robust class ofhistone deacetylases that inhibit these enzymes by virtue of hydroxamatefunctionality that binds cationic zinc within the active sites of theseenzymes. Exemplary inhibitors include trichostatin A, described above,as well as Vorinostat (N-hydroxy-N′-phenyl-octanediamide, described inMarks et al., Nature Biotechnology 25, 84 to 90 (2007); Stenger,Community Oncology 4, 384-386 (2007), the disclosures of which areincorporated by reference herein). Other histone deacetylase inhibitorsinclude Panobinostat, described in Drugs of the Future 32(4): 315-322(2007), the disclosure of which is incorporated herein by reference.

Additional examples of hydroxamic acid inhibitors of histonedeacetylases include the compounds shown below, described in Bertrand,European Journal of Medicinal Chemistry 45:2095-2116 (2010), thedisclosure of which is incorporated herein by reference:

Other histone deacetylase inhibitors that do not contain a hydroxamatesubstituent have also been developed, including Valproic acid(Gottlicher et al. EMBO Journal 20: 6969 (2001) and Mocetinostat(N-(2-Aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide,described in Balasubramanian et al. Cancer Letters 280: 211 (2009), thedisclosures of each of which are incorporated herein by reference. Othersmall molecule inhibitors that exploit chemical functionality distinctfrom a hydroxamate include those described in Bertrand, European Journalof Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which isincorporated herein by reference. Other small molecule inhibitors thatexploit chemical functionality distinct from a hydroxamate to inhibithistone deacetylases include those shown below.

Additional examples of chemical modulators of histone acetylation usefulwith the compositions and methods of the invention to expand, enrich,and maintain the hematopoietic stem cell functional potential ofhematopoietic stem cells include modulators of HDAC1, HDAC2, HDAC3,HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, Sirt1, Sirt2, and/orHAT, such as butyrylhydroxamic acid, M344, LAQ824 (Dacinostat), AR-42,Belinostat (PXD101), CUDC-101, Scriptaid, Sodium Phenylbutyrate,Tasquinimod, Quisinostat (JNJ-26481585), Pracinostat (SB939), CUDC-907,Entinostat (MS-275), Mocetinostat (MGCD0103), Tubastatin A HCl,PCI-34051, Droxinostat, PCI-24781 (Abexinostat), RGFP966, Rocilinostat(ACY-1215), CI994 (Tacedinaline), Tubacin, RG2833 (RGFP109),Resminostat, Tubastatin A, BRD73954, BG45, 4SC-202, CAY10603, LMK-235,Nexturastat A, TMP269, HPOB, Cambinol, and Anacardic Acid.

Antibodies and Other Therapeutic Proteins

Antibodies represent a region of chemical space that is uniquely suitedto target extracellular protein-protein interactions, such asreceptor-ligand interactions. These agents possess the large molecularvolume that is beneficial to inhibiting interactions that featureresidues that contribute favorably to the free energy of the interactiondispersed over vast surfaces rather than confined to a shallow crevice.Antibody agents possess a large molecular volume that is beneficial forinhibiting interactions that occur over vast surfaces rather than withinshallow crevice. Inhibitory antibodies may function by binding anextracellular receptor in such a way that sterically precludesinteraction of the receptor with the cognate ligand and thus maintainsthe receptor in an inactive conformation. For instance, inhibitoryantibodies capable of attenuating TGFβ receptor activity includeLerdelimumab, and an antibody that binds the TGFβ receptor type II.Other examples include GC-1008, an antibody that binds and antagonizesall isoforms of human TGFβ, as well as ID11, an antibody that binds allisoforms of murine TGFβ. These antibodies are described in detail, e.g.,in U.S. Pat. No. 8,603,818, the disclosure of which is incorporatedherein by reference.

Antagonist antibodies have also been developed that inhibit β-cateninphosphorylation by virtue of propagating the Wnt signal transductioncascade. Such antibodies may bind the Wnt receptors including Frizzledand LRP family proteins and trigger concomitant conformational changesthat stimulate the propagation of the Wnt signaling pathway, whichincludes distinct molecular events that inhibit β-cateninphosphorylation by GKS3. For instance, the antibody 1D9 has beendeveloped as an agonist of Wnt signal transduction, and is described indetail in US 2014/0044717, the disclosure of which is incorporatedherein by reference.

Other classes of therapeutic proteins may additionally be used forinhibiting biological processes for expanding, enriching, andmaintaining the hematopoietic stem cell functional potential ofhematopoietic stem cells. Endogenous proteins that modulate signaltransduction events may be used to attenuate these events ex vivo,thereby leveraging the natural affinity of these proteins for theircognate ligands in order to antagonize or stimulate importantprotein-protein interactions. For instance, a variety of proteins thatantagonize the TGFβ signaling cascade can be used to this end, includingDecorin, an extracellular matrix proteoglycan that negatively regulatesTGFβ activity, as well as Lefty1, Lefty2, Follistatin, Noggin, Chordin,Cerberus, Germlin, Inhibin, Cystatin C, Recombinant Mouse Lefty-1 (anACVR2B inhibitor), as well as the Smad proteins Smad6 and Smad7, whichserve to prevent the phosphorylation of the R-Smad proteins or recruitubiquitin ligases to the TGFβ receptor type I so as to promote thedegradation of the receptor. These proteins are described in detail inU.S. Pat. No. 8,298,825, the disclosure of which is incorporated byreference herein.

Another modulator of TGFβ signaling that may be used in conjunction withthe compositions and methods of the invention to expand, enrich, andmaintain the hematopoietic stem cell functional potential ofhematopoietic stem cells is Recombinant Amphibian TGF-ß5 (an ACVR2A,ACVR2B, TGFβ RII activator).

In addition to the negative feedback proteins described above, proteinscapable of inducing Wnt signaling so as to inhibit β-cateninphosphorylation have also been described. For instance, Rspondin (roofplate-specific spondin) proteins are also known to activate β-cateninsignaling. Rspondin proteins do not bear sequence similarity to Wntproteins and appear to potentiate Wnt signaling through aFrizzled-independent mechanism. This protein is described in detail inKazanskaya. O., et al., Dev. Cell 7, 525-534 (2004), the disclosure ofwhich is incorporated herein by reference.

Interfering RNA

RNA interference (RNAi) represents an inhibitory modality that exploitsthe ability of antagonistic RNA (e.g., double-stranded RNA containing anoligonucleotide capable of complementary hydrogen bond-mediated basepairing with an endogenous mRNA sequence) to attenuate intracellulargene expression. Mechanistically, this phenomenon often operates by wayof degradation of the complementary mRNA or by steric inhibition ofribosome formation at the mRNA transcript. Long stretches of dsRNAs areoften cleaved in the cytoplasm of a eukaryotic cell into short 21-25nucleotide small interfering RNAs, known as siRNAs, by the ribonucleaseknown Dicer. These siRNAs subsequently assemble with protein componentsinto an RNA-induced silencing complex (RISC), unwinding in the process.Activated RISC then binds to complementary transcript by base pairinginteractions between the siRNA antisense strand and the mRNA. The boundmRNA is cleaved and sequence specific degradation of mRNA results ingene silencing. The molecular events that underlie RISC-mediated genesilencing are described, for example, in U.S. Pat. No. 6,506,559; Fireet al., Nature 391(19):306-311 (1998); Timmons et al., Nature 395:854(1998); Montgomery et al., TIG 14 (7):255-258 (1998); David R. Engelke,Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press,Eagleville, Pa. (2003); and Gregory J. Hannon, Ed., RNAi A Guide to GeneSilencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2003), the disclosures of which are incorporated herein by reference.

Significantly, siRNA molecules useful with the compositions and methodsof the invention need not be limited to those molecules containing onlyRNA, but may include, for example, chemically modified nucleotides andnon-nucleotides that effect RNA interference, as well as moleculeswherein a ribose sugar is replaced with another sugar molecule or analogthereof. Moreover, a non-natural linkage between nucleotide residues canbe used, such as a phosphorothioate linkage, which is less susceptibleto phosphodiesterase-mediated degradation. The RNA oligonucleotidesuseful with the compositions and methods of the invention may also bederivatized with a reactive functional group or a reporter group, suchas a fluorophore. Particularly useful derivatives are modified at aterminus or termini of an RNA strand, typically the 3′ terminus of thesense strand. For example, the 2′-hydroxyl at the 3′ terminus can bereadily and selectively derivatized with a variety of groups by standardnucleophilic substitution techniques known in the art. Other useful RNAderivatives incorporate nucleotides having modified carbohydratemoieties, such as 2′O-alkylated residues or 2′-O-methyl ribosylderivatives and 2′-O-fluoro ribosyl derivatives that impartoligonucleotides with enhanced structural stability. The nucleobases ofsiRNAs may also be chemically modified. For example, halogenated bases,such as 5-bromouracil and 5-iodouracil can be incorporated into a siRNAmolecule so as to modulate the strength of hydrogen bonding interactionswith target mRNA. The nucleobases may also be strategically alkylated;for instance, 7-methylguanosine can be incorporated in place of aguanosine residue. Non-natural bases that promote inhibition of targetgene expression may also be incorporated into interfering RNAs. OthersiRNA modifications include 2′-deoxy-2′-fluorouridine or locked nucleicacid (LNA) nucleotides and RNA duplexes containing either phosphodiesteror varying numbers of phosphorothioate linkages. Such modifications aredescribed, for example, in Braasch et al., Biochemistry 42: 7967-7975(2003), the disclosure of which is incorporated herein by reference.

Synthetic siRNA molecules, can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA molecule canbe chemically synthesized or recombinantly produced using methods knownin the art, such as using appropriately protected ribonucleosidephosphoramidites and conventional solid-phase oligonucleotide synthesis(see, e.g., Elbashir et al. Nature 411:494 (2001); Elbashir et al. Genes& Development 15:188 (2001); Harborth. et al. Journal of Cell Science114:4557 (2001); Masters et al. Proceeding of the National Academy ofSciences USA 98:8012 (2001); and Tuschl et al. Genes & Development13:3191 (1999), the disclosures of which are incorporated herein byreference). In addition, dsRNAs can be expressed as stem loop structuresencoded by plasmid vectors, retroviruses and lentiviruses (see, e.g.,Paddison et al. Genes and Development 16:948 (2002); McManus et al. RNA8:842 (2002); Paul et al. Nature Biotechnology 20:505 (2002); Miyagishiet al. Nature Biotechnology 20:497 (2002); Sui et al. Proceedings of theNational Academy of Sciences USA 99:5515 (2002); Brummelkamp et al.Cancer Cell 2:243 (2002); Lee et al. Nature Biotechnology 20:500 (2002);Yu et al. Proceedings of the National Academy of Sciences USA 99:6047(2002); Zeng et al. Molecular Cell 9:1327 (2002); Rubinson et al. NatureGenetics 33:401 (2003); Stewart et al. RNA 9:493 (2003), the disclosuresof each of which are incorporated herein by reference).

A variety of inhibitory agents that operate by a mechanism of RNAinterference have been developed to antagonize the biological processesdescribed by the methods of the invention. For instance, TGFβ receptortype II siRNA polynucleotides have been reported that are derived fromthe human TGFβRII sequence (Genbank Accession Number: M85079). The siRNAduplex sequences below were developed against particular targetsequences within the TGFβ receptor type II gene and have been used toknock down expression of the receptor in a variety of whole cell models.These siRNA sequences are described in detail in U.S. Pat. No.8,067,389, the disclosure of which is incorporated herein by reference.Other oligonucleotide-based modulators of TGFβ signaling, such as siRNAsand antisense oligonucleotides, are described in U.S. Pat. Nos.5,731,424; 6,124,449; US 2008/0015161; US 2006/0229266; US 2004/0006030;US 2005/0227936; and US 2005/0287128, the disclosures of each of whichare incorporated herein by reference. siRNAs useful for targeting TGFβRor ALK5 expression can be readily designed and tested. A database ofsiRNA sequences and a predictor of siRNA sequences has been established(Chalk et al. (Nucleic Acids Research 33: D131 (2005). This database canbe used to predict the thermodynamic parameters of a particularsiRNA-target mRNA interaction, as well as to evaluate the propensity ofdesigned siRNA sequences for off-target interactions. The database isavailable as an electronic resource at www.siRNA.cgb.ki.se.

Target Sequence 5′ to 3′ and Nucleotide number siRNA duplex Nt 529UCCUGCAUGAGCAACUGCAdTdT AATCCTGCATGAGCAACTGCA dTdTAGGACGUACUCGUUGACGU(SEQ ID NO: 1) (SEQ ID NOS: 2 and 3) Nt 1113 GGCCAAGCUGAAGCAGAACdTdTAAGGCCAAGCTGAAGCAGAAC dTdTCCGGUUCGACUUCGUCUUG (SEQ ID NO: 4) (SEQ IDNOS: 5 and 6) Nt 1253 GCAUGAGAACAUACUCCAGdTdT AGCATGAGAACATACTCCAGdTdTCGUACUCUUGUAUGAGGUC (SEQ ID NO: 7) (SEQ ID NO: 8 and 9) Nt 948GACGCGGAAGCUCAUGGAGdTdT AAGACGCGGAAGCTCATGGAG dTdTCUGCGCCUUCGAGUACCUC(SEQ ID NO: 10) (SEQ ID NO: 11 and 12)Conformationally Constrained Peptides

Peptide-based therapeutics represent an emerging class of compoundsuseful for the inhibition of protein-protein interactions that haveoften been intractable to inhibition by other means. Conformationallyrestricted peptides offer particular advantages for therapeuticapplications, as these compounds often exhibit enhanced target affinityand selectivity by virtue of presenting structurally pre-organizedepitopes in which a particular pharmacophore is spatially predisposedfor interacting with a protein of interest. Constrained peptides oftenfeature the additional benefits of enhanced protease resistivityrelative to their unconstrained (e.g., linear) counterparts byrestricting the access of proteases to internal amide bonds. The cellpenetrating capabilities of these compounds are also frequently higherthan those of linear peptides due to the sequestration of hydrogen bonddonors and acceptors from aqueous solvent. Exemplary constrained peptideinhibitors useful with the compositions and methods of the inventioninclude olefin “stapled” peptides, which often feature alpha helicesthat have been structurally rigidified by insertion of a covalentcross-link between residues on the same face of the helix. This class ofconstrained peptides is described, e.g., in Walensky et al. Journal ofMedicinal Chemistry 57:6275 (2014), the disclosure of which isincorporated herein by reference. Stapled peptide inhibitors ofβ-catenin phosphorylation have been developed that function bydisrupting the Axin/β-catenin interaction. Axin serves to anchorβ-catenin to a protein complex that includes GSK3, and the associationhas been shown to be mediated by the insertion of an alpha helicalregion of Axin into a shallow pocket at the β-catenin surface. A stapledpeptide of the sequence Ac-PQR₈ILDQHVS₅RVMK-NH₂ (SEQ ID NO: 13) has beenreported that is structurally restricted to an alpha helicalconformation by virtue of an olefinic cross-link at residues R₈((S)-α-(7-octenyl)alanine) and S₅ ((S)-α-(4-pentenyl)alanine) (see,e.g., Cui et al. Cell Research 23: 581 (2013), the disclosure of whichis incorporated herein by reference). This peptide competes with Axinfor binding at the surface of β-catenin and serves to liberate theprotein from the GSK3-containing complex, thus increasing the nuclearconcentration of this transcription factor.

Constrained peptides have also been developed by virtue of covalentcyclization between the N- and C-termini. Exemplary inhibitors of thisclass include the depsipeptides, which feature lactone moieties thatrender these peptide macrocyclic. Depsipeptide inhibitors useful withthe compositions and methods of the invention include inhibitors ofhistone deacetylases, such as Romidepsin (also referred to as istodax;structure shown below), which is described in, e.g., Vinodhkumar et al.,Biomedicine & Pharmacotherapy 62:85-93 (2008), the disclosure of whichis incorporated by reference herein.

Additional examples of depsipeptide inhibitors of histone deacetylasesinclude Apicidin, described in Bertrand, European Journal of MedicinalChemistry 45:2095-2116 (2010), the disclosure of which is incorporatedherein by reference.

Combinations of Compounds Useful with the Compositions and Methods ofthe Invention

Embodiments of the compositions and methods of the invention may containcombinations of any of the above-described compounds may be used, e.g.,for the expansion, enrichment, and maintenance of hematopoietic stemcell functional potential of a population of hematopoietic stem cells.Particular combination of compounds useful in conjunction with thecompositions and methods of the inventions are specified in Tables 1-10,below.

TABLE 1 2-Component Combination of pathway inhibitors 3-ComponentCombination of pathway inhibitors Histone methylation TGFβ signalinginhibitor Histone methylation TGFβ signaling inhibitor p38 signalinginhibitor inhibitor inhibitor Histone methylation p38 signalinginhibitor Histone methylation TGFβ signaling inhibitor Wnt signalingagonist inhibitor inhibitor Histone methylation Wnt signaling agonistHistone methylation TGFβ signaling inhibitor Histone acetylationinhibitor inhibitor inhibitor Histone methylation Histone acetylationTGFβ signaling inhibitor p38 signaling inhibitor Wnt signaling agonistinhibitor inhibitor TGFβ signaling inhibitor p38 signaling inhibitorTGFβ signaling inhibitor p38 signaling inhibitor Histone acetylationinhibitor TGFβ signaling inhibitor Wnt signaling agonist p38 signalinginhibitor Histone methylation Wnt signaling agonist inhibitor TGFβsignaling inhibitor Histone acetylation p38 signaling inhibitor Histonemethylation Histone acetylation inhibitor inhibitor inhibitor p38signaling inhibitor Wnt signaling agonist Wnt signaling agonist Histonemethylation Histone acetylation inhibitor inhibitor p38 signalinginhibitor Histone acetylation TGFβ signaling inhibitor Wnt signalingagonist Histone acetylation inhibitor inhibitor Histone acetylation Wntsignaling agonist p38 signaling inhibitor Wnt signaling agonist Histoneacetylation inhibitor inhibitor Histone demethylation TGFβ signalinginhibitor Histone demethylation TGFβ signaling inhibitor p38 signalinginhibitor inhibitor inhibitor Histone demethylation p38 signalinginhibitor Histone demethylation TGFβ signaling inhibitor Wnt signalingagonist inhibitor inhibitor Histone demethylation Wnt signaling agonistHistone demethylation TGFβ signaling inhibitor Histone acetylationinhibitor inhibitor inhibitor Histone demethylation Histone acetylationHistone demethylation TGFβ signaling inhibitor Histone deacetylationinhibitor inhibitor inhibitor Histone demethylation Histonedeacetylation Histone demethylation TGFβ signaling inhibitor Histonemethylation inhibitor inhibitor inhibitor inhibitor Histonedemethylation Histone methylation Histone demethylation p38 signalinginhibitor Wnt signaling agonist inhibitor inhibitor inhibitor Histonedeacetylation TGFβ signaling inhibitor Histone demethylation p38signaling inhibitor Histone acetylation inhibitor inhibitor inhibitorHistone deacetylation p38 signaling inhibitor Histone demethylation p38signaling inhibitor Histone deacetylation inhibitor inhibitor inhibitorHistone deacetylation Wnt signaling agonist Histone demethylation p38signaling inhibitor Histone methylation inhibitor inhibitor inhibitorHistone deacetylation Histone acetylation Histone demethylation Wntsignaling agonist Histone acetylation inhibitor inhibitor inhibitorinhibitor Histone deacetylation Histone methylation Histonedemethylation Wnt signaling agonist Histone deacetylation inhibitorinhibitor inhibitor inhibitor Histone demethylation Wnt signalingagonist Histone methylation inhibitor inhibitor Histone demethylationHistone acetylation Histone deacetylation inhibitor inhibitor inhibitorHistone demethylation Histone methylation Histone acetylation inhibitorinhibitor inhibitor Histone deacetylation TGFβ signaling inhibitor p38signaling inhibitor inhibitor Histone deacetylation TGFβ signalinginhibitor Wnt signaling agonist inhibitor Histone deacetylation TGFβsignaling inhibitor Histone acelylation inhibitor inhibitor Histonedeacetylation TGFβ signaling inhibitor Histone methylation inhibitorinhibitor Histone deacetylation p38 signaling inhibitor Wnt signalingagonist inhibitor Histone deacetylation p38 signaling inhibitor Histoneacetylation inhibitor inhibitor Histone deacetylation p38 signalinginhibitor Histone methylation inhibitor inhibitor Histone deacetylationWnt signaling agonist Histone acetylation inhibitor inhibitor Histonedeacetylation Wnt signaling agonist Histone methylation inhibitorinhibitor Histone deacetylation Histone acetylation Histone methylationinhibitor inhibitor inhibitor Histone deacetylation Histone methylationHistone demethylation inhibitor inhibitor inhibitor

TABLE 2 4-Component Combination of pathway inhibitors Histonemethylation Histone demethylation TGFβ signaling inhibitor p38 signalinginhibitor inhibitor inhibitor Histone methylation Histone demethylationTGFβ signaling inhibitor Wnt signaling agonist inhibitor inhibitorHistone methylation Histone demethylation TGFβ signaling inhibitorHistone acetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor Histone deacetylationinhibitor inhibitor inhibitor Histone methylation Histone demethylationp38 signaling inhibitor Wnt signaling agonist inhibitor inhibitorHistone methylation Histone demethylation p38 signaling inhibitorHistone acetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation p38 signaling inhibitor Histone deacetylationinhibitor inhibitor inhibitor Histone methylation Histone demethylationWnt signaling agonist Histone acetylation inhibitor inhibitor inhibitorHistone methylation Histone demethylation Wnt signaling agonist Histonedeacetylation inhibitor inhibitor inhibitor Histone methylation Histonedemethylation Histone acelylation Histone deacetylation inhibitorinhibitor inhibitor inhibitor Histone methylation TGFβ signalinginhibitor p38 signaling inhibitor Wnt signaling agonist inhibitorHistone methylation TGFβ signaling inhibitor p38 signaling inhibitorHistone acetylation inhibitor inhibitor Histone methylation TGFβsignaling inhibitor p38 signaling inhibitor Histone deacetylationinhibitor inhibitor Histone methylation TGFβ signaling inhibitor Wntsignaling agonist Histone acetylation inhibitor inhibitor Histonemethylation TGFβ signaling inhibitor Wnt signaling agonist Histonedeacetylation inhibitor inhibitor Histone methylation TGFβ signalinginhibitor Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor Histone methylation p38 signaling inhibitor Wnt signalingagonist Histone acetylation inhibitor inhibitor Histone methylation p38signaling inhibitor Wnt signaling agonist Histone deacetylationinhibitor inhibitor Histone methylation p38 signaling inhibitor Histoneacetylation Histone deacetylation inhibitor inhibitor inhibitor Histonemethylation Wnt signaling agonist Histone acetylation Histonedeacetylation inhibitor inhibitor inhibitor Histone demethylation TGFβsignaling inhibitor p38 signaling inhibitor Wnt signaling agonistinhibitor Histone demethylation TGFβ signaling inhibitor p38 signalinginhibitor Histone acetylation inhibitor inhibitor Histone demethylationTGFβ signaling inhibitor p38 signaling inhibitor Histone deacetylationinhibitor inhibitor Histone demethylation TGFβ signaling inhibitor Wntsignaling agonist Histone acetylation inhibitor inhibitor Histonedemethylation TGFβ signaling inhibitor Wnt signaling agonist Histonedeacetylation inhibitor inhibitor Histone demethylation TGFβ signalinginhibitor Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor Histone demethylation p38 signaling inhibitor Wnt signalingagonist Histone acetylation inhibitor inhibitor Histone demethylationp38 signaling inhibitor Wnt signaling agonist Histone deacetylationinhibitor inhibitor Histone demethylation p38 signaling inhibitorHistone acetylation Histone deacetylation inhibitor inhibitor inhibitorHistone demethylatlon Wnt signaling agonist Histone acetylation Histonedeacetylation inhibitor inhibitor inhibitor TGFβ signaling inhibitor p38signaling inhibitor Wnt signaling agonist Histone acetylation inhibitorTGFβ signaling inhibitor p38 signaling inhibitor Wnt signaling agonistHistone deacetylation inhibitor TGFβ signaling inhibitor p38 signalinginhibitor Histone acetylation Histone deacetylation inhibitor inhibitorTGFβ signaling inhibitor Wnt signaling agonist Histone acetylationHistone deacetylation inhibitor inhibitor p38 signaling inhibitor Wntsignaling agonist Histone acetylation Histone deacetylation inhibitorinhibitor

TABLE 3 5-Component Combination of pathway inhibitors Histonemethylation Histone demethylation TGFβ signaling inhibitor p38 signalinginhibitor Wnt signaling agonist inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor p38 signaling inhibitorHistone acetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor p38 signaling inhibitorHistone deacetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor Wnt signaling agonistHistone acetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor Wnt signaling agonistHistone deacetylation inhibitor inhibitor inhibitor Histone methylationHistone demethylation TGFβ signaling inhibitor Histone acetylationHistone deacetylation inhibitor inhibitor inhibitor inhibitor Histonemethylation Histone demethylation p38 signaling inhibitor Wnt signalingagonist Histone acetylation inhibitor inhibitor inhibitor Histonemethylation Histone demethylation p38 signaling inhibitor Wnt signalingagonist Histone deacetylation inhibitor inhibitor inhibitor Histonemethylation Histone demethylation p38 signaling inhibitor Histoneacetylation Histone deacetylation inhibitor inhibitor inhibitorinhibitor Histone methylation Histone demethylation Wnt signalingagonist Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor inhibitor Histone methylation TGFβ signaling inhibitor p38signaling inhibitor Wnt signaling agonist Histone acetylation inhibitorinhibitor Histone methylation TGFβ signaling inhibitor p38 signalinginhibitor Wnt signaling agonist Histone deacetylation inhibitorinhibitor Histone methylation TGFβ signaling inhibitor p38 signalinginhibitor Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor Histone methylation TGFβ signaling inhibitor Wnt signalingagonist Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor Histone methylation p38 signaling inhibitor Wnt signalingagonist Histone acetylation Histone deacetylation inhibitor inhibitorinhibitor Histone demethylation TGFβ signaling inhibitor p38 signalinginhibitor Wnt signaling agonist Histone acetylation inhibitor inhibitorHistone demethylation TGFβ signaling inhibitor p38 signaling inhibitorWnt signaling agonist Histone deacetylation inhibitor inhibitor Histonedemethylation TGFβ signaling inhibitor p38 signaling inhibitor Histoneacetylation Histone deacetylation inhibitor inhibitor inhibitor Histonedemethylation TGFβ signaling inhibitor Wnt signaling agonist Histoneacetylation Histone deacetylation inhibitor inhibitor inhibitor Histonedemethylation p38 signaling inhibitor Wnt signaling agonist Histoneacetylation Histone deacetylation inhibitor inhibitor inhibitor TGFβsignaling inhibitor p38 signaling inhibitor Wnt signaling agonistHistone acetylation Histone deacetylation inhibitor inhibitor

TABLE 4 2-Component Combination 3-Component Combination LSD1 TGFβ LSD1TGFβ p38 inhibitor inhibitor inhibitor inhibitor inhibitor LSD1 p38 LSD1TGFβ Wnt inhibitor inhibitor inhibitor inhibitor agonist LSD1 Wnt LSD1TGFβ HDAC inhibitor agonist inhibitor inhibitor inhibitor LSD1 HDAC TGFβp38 Wnt inhibitor inhibitor inhibitor inhibitor agonist TGFβ p38 TGFβp38 HDAC inhibitor inhibitor inhibitor inhibitor inhibitor TGFβ Wnt p38LSD1 Wnt inhibitor agonist inhibitor inhibitor agonist TGFβ HDAC p38LSD1 HDAC inhibitor inhibitor inhibitor inhibitor inhibitor p38 Wnt WntLSD1 HDAC inhibitor agonist agonist inhibitor inhibitor p38 HDAC TGFβWnt HDAC inhibitor inhibitor inhibitor agonist inhibitor HDAC Wnt p38Wnt HDAC inhibitor agonist inhibitor agonist inhibitor

TABLE 5 4-Component Combination of Target Inhibitors LSD1 TGFβ p38 Wntinhibitor inhibitor inhibitor agonist LSD1 TGFβ p38 HDAC inhibitorinhibitor inhibitor inhibitor TGFβ Wnt p38 HDAC inhibitor agonistinhibitor inhibitor p38 LSD1 Wnt HDAC inhibitor inhibitor agonistinhibitor Wnt LSD1 TGFβ HDAC agonist inhibitor inhibitor inhibitor

TABLE 6 5-Component Combination of Target Inhibitors LSD1 TGFβ p38 WntHDAC inhibitor inhibitor inhibitor agonist inhibitor

TABLE 7 No. 2-Component Combination 1 LSD1 inhibitor IV RN ALK5inhibitor II 2 LSD1 inhibitor IV RN LY364947 3 LSD1 inhibitor IV RNA83-01 4 LSD1 inhibitor IV RN DMH1 5 LSD1 inhibitor II S2101 ALK5inhibitor II 6 LSD1 inhibitor II S2101 LY364947 7 LSD1 inhibitor IIS2101 A83-01 8 LSD1 inhibitor II S2101 DMH1 9 LSD1 inhibitor LSD1-C76ALK5 inhibitor II 10 LSD1 inhibitor LSD1-C76 LY364947 11 LSD1 inhibitorLSD1-C76 A83-01 12 LSD1 inhibitor LSD1-C76 DMH1 13 LSD1 inhibitor IIICBB1007 ALK5 inhibitor II 14 LSD1 inhibitor III CBB1007 LY364947 15 LSD1inhibitor III CBB1007 A83-01 16 LSD1 inhibitor III CBB1007 DMH1 17 LSD1inhibitor I ALK5 inhibitor II 18 LSD1 inhibitor I LY364947 19 LSD1inhibitor I A83-01 20 LSD1 inhibitor I DMH1 21 Tranylcypromine ALK5inhibitor II 22 Tranylcypromine LY364947 23 Tranylcypromine A83-01 24Tranylcypromine DMH1 25 LSD1 inhibitor IV RN SB203580 26 LSD1 inhibitorII S2101 SB203580 27 LSD1 inhibitor LSD1-C76 SB203580 28 LSD1 inhibitorIII CBB1007 SB203580 29 LSD1 inhibitor I SB203580 30 TranylcypromineSB203580 31 LSD1 inhibitor IV RN CHIR99021 32 LSD1 inhibitor II S2101CHIR99021 33 LSD1 inhibitor LSD1-C76 CHIR99021 34 LSD1 inhibitor IIICBB1007 CHIR99021 35 LSD1 inhibitor I CHIR99021 36 TranylcypromineCHIR99021 37 LSD1 inhibitor IV RN Trichostatin A 38 LSD1 inhibitor IV RNIstodax 39 LSD1 inhibitor II S2101 Trichostatin A 40 LSD1 inhibitor IIS2101 Istodax 41 LSD1 inhibitor LSD1-C76 Trichostatin A 42 LSD1inhibitor LSD1-C76 Istodax 43 LSD1 inhibitor III CBB1007 Trichostatin A44 LSD1 inhibitor III CBB1007 Istodax 45 LSD1 inhibitor I Trichostatin A46 LSD1 inhibitor I Istodax 47 Tranylcypromine Trichostatin A 48Tranylcypromine Istodax 49 ALK5 inhibitor II SB203580 50 LY364947SB203580 51 A83-01 SB203580 52 DMH1 SB203580 53 ALK5 inhibitor IICHIR99021 54 LY364947 CHIR99021 55 A83-01 CHIR99021 56 DMH1 CHIR99021 57ALK5 inhibitor II Trichostatin A 58 ALK5 inhibitor II istodax 59LY364947 Trichostatin A 60 LY364947 istodax 61 A83-01 Trichostatin A 62A83-01 istodax 63 DMH1 Trichostatin A 64 DMH1 istodax 65 SB203580CHIR99021 66 Trichostatin A SB203580 67 istodax SB203580 68 TrichostatinA CHIR99021 69 istodax CHIR99021

TABLE 8 No. 3-Component Combination 1 LSD1 inhibitor IV RN ALK5inhibitor II SB203580 2 LSD1 inhibitor IV RN LY364947 SB203580 3 LSD1inhibitor IV RN A83-01 SB203580 4 LSD1 inhibitor IV RN DMH1 SB203580 5LSD1 inhibitor II S2101 ALK5 inhibitor II SB203580 6 LSD1 inhibitor IIS2101 LY364947 SB203580 7 LSD1 inhibitor II S2101 A83-01 SB203580 8 LSD1inhibitor II S2101 DMH1 SB203580 9 LSD1 inhibitor LSD1-C76 ALK5inhibitor II SB203580 10 LSD1 inhibitor LSD1-C76 LY364947 SB203580 11LSD1 inhibitor LSD1-C76 A83-01 SB203580 12 LSD1 inhibitor LSD1-C76 DMH1SB203580 13 LSD1 inhibitor III CBB1007 ALK5 inhibitor II SB203580 14LSD1 inhibitor III CBB1007 LY364947 SB203580 15 LSD1 inhibitor IIICBB1007 A83-01 SB203580 16 LSD1 inhibitor III CBB1007 DMH1 SB203580 17LSD1 inhibitor I ALK5 inhibitor II SB203580 18 LSD1 inhibitor I LY364947SB203580 19 LSD1 inhibitor I A83-01 SB203580 20 LSD1 inhibitor I DMH1SB203580 21 Tranylcypromine ALK5 inhibitor II SB203580 22Tranylcypromine LY364947 SB203580 23 Tranylcypromine A83-01 SB203580 24Tranylcypromine DMH1 SB203580 25 LSD1 inhibitor IV RN ALK5 inhibitor IICHIR99021 26 LSD1 inhibitor IV RN LY364947 CHIR99021 27 LSD1 inhibitorIV RN A83-01 CHIR99021 28 LSD1 inhibitor IV RN DMH1 CHIR99021 29 LSD1inhibitor II S2101 ALK5 inhibitor II CHIR99021 30 LSD1 inhibitor IIS2101 LY364947 CHIR99021 31 LSD1 inhibitor II S2101 A83-01 CHIR99021 32LSD1 inhibitor II S2101 DMH1 CHIR99021 33 LSD1 inhibitor LSD1-C76 ALK5inhibitor II CHIR99021 34 LSD1 inhibitor LSD1-C76 LY364947 CHIR99021 35LSD1 inhibitor LSD1-C76 A83-01 CHIR99021 36 LSD1 inhibitor LSD1-C76 DMH1CHIR99021 37 LSD1 inhibitor III CBB1007 ALK5 inhibitor II CHIR99021 38LSD1 inhibitor III CBB1007 LY364947 CHIR99021 39 LSD1 inhibitor IIICBB1007 A83-01 CHIR99021 40 LSD1 inhibitor III CBB1007 DMH1 CHIR99021 41LSD1 inhibitor I ALK5 inhibitor II CHIR99021 42 LSD1 inhibitor ILY364947 CHIR99021 43 LSD1 inhibitor I A83-01 CHIR99021 44 LSD1inhibitor I DMH1 CHIR99021 45 Tranylcypromine ALK5 inhibitor IICHIR99021 46 Tranylcypromine LY364947 CHIR99021 47 TranylcypromineA83-01 CHIR99021 48 Tranylcypromine DMH1 CHIR99021 49 LSD1 inhibitor IVRN ALK5 inhibitor II Trichostatin A 50 LSD1 inhibitor IV RN LY364947Trichostatin A 51 LSD1 inhibitor IV RN A83-01 Trichostatin A 52 LSD1inhibitor IV RN DMH1 Trichostatin A 53 LSD1 inhibitor II S2101 ALK5inhibitor II Trichostatin A 54 LSD1 inhibitor II S2101 LY364947Trichostatin A 55 LSD1 inhibitor II S2101 A83-01 Trichostatin A 56 LSD1inhibitor II S2101 DMH1 Trichostatin A 57 LSD1 inhibitor LSD1-C76 ALK5inhibitor II Trichostatin A 58 LSD1 inhibitor LSD1-C76 LY364947Trichostatin A 59 LSD1 inhibitor LSD1-C76 A83-01 Trichostatin A 60 LSD1inhibitor LSD1-C76 DMH1 Trichostatin A 61 LSD1 inhibitor III CBB1007ALK5 inhibitor II Trichostatin A 62 LSD1 inhibitor III CBB1007 LY364947Trichostatin A 63 LSD1 inhibitor III CBB1007 A83-01 Trichostatin A 64LSD1 inhibitor III CBB1007 DMH1 Trichostatin A 65 LSD1 inhibitor I ALK5inhibitor II Trichostatin A 66 LSD1 inhibitor I LY364947 Trichostatin A67 LSD1 inhibitor I A83-01 Trichostatin A 68 LSD1 inhibitor I DMH1Trichostatin A 69 Tranylcypromine ALK5 inhibitor II Trichostatin A 70Tranylcypromine LY364947 Trichostatin A 71 Tranylcypromine A83-01Trichostatin A 72 Tranylcypromine DMH1 Trichostatin A 73 LSD1 inhibitorIV RN ALK5 inhibitor II Istodax 74 LSD1 inhibitor IV RN LY364947 Istodax75 LSD1 inhibitor IV RN A83-01 Istodax 76 LSD1 inhibitor IV RN DMH1Istodax 77 LSD1 inhibitor II S2101 ALK5 inhibitor II Istodax 78 LSD1inhibitor II S2101 LY364947 Istodax 79 LSD1 inhibitor II S2101 A83-01Istodax 80 LSD1 inhibitor II S2101 DMH1 Istodax 81 LSD1 inhibitorLSD1-C76 ALK5 inhibitor II Istodax 82 LSD1 inhibitor LSD1-C76 LY364947Istodax 83 LSD1 inhibitor LSD1-C76 A83-01 Istodax 84 LSD1 inhibitorLSD1-C76 DMH1 Istodax 85 LSD1 inhibitor III CBB1007 ALK5 inhibitor IIIstodax 86 LSD1 inhibitor III CBB1007 LY364947 Istodax 87 LSD1 inhibitorIII CBB1007 A83-01 Istodax 88 LSD1 inhibitor III CBB1007 DMH1 Istodax 89LSD1 inhibitor I ALK5 inhibitor II Istodax 90 LSD1 inhibitor I LY364947Istodax 91 LSD1 inhibitor I A83-01 Istodax 92 LSD1 inhibitor I DMH1Istodax 93 Tranylcypromine ALK5 inhibitor II Istodax 94 TranylcypromineLY364947 Istodax 95 Tranylcypromine A83-01 Istodax 96 TranylcypromineDMH1 istodax 97 ALK5 inhibitor II SB203580 CHIR99021 98 LY364947SB203580 CHIR99021 99 A83-01 SB203580 CHIR99021 100 DMH1 SB203580CHIR99021 101 ALK5 inhibitor II SB203580 Trichostatin A 102 LY364947SB203580 Trichostatin A 103 A83-01 SB203580 Trichostatin A 104 DMH1SB203580 Trichostatin A 105 ALK5 inhibitor II SB203580 istodax 106LY364947 SB203580 istodax 107 A83-01 SB203580 istodax 108 DMH1 SB203580istodax 109 LSD1 inhibitor IV RN SB203580 CHIR99021 110 LSD1 inhibitorII S2101 SB203580 CHIR99021 111 LSD1 inhibitor LSD1-C76 SB203580CHIR99021 112 LSD1 inhibitor III CBB1007 SB203580 CHIR99021 113 LSD1inhibitor I SB203580 CHIR99021 114 Tranylcypromine SB203580 CHIR99021115 LSD1 inhibitor IV RN SB203580 Trichostatin A 116 LSD1 inhibitor IIS2101 SB203580 Trichostatin A 117 LSD1 inhibitor LSD1-C76 SB203580Trichostatin A 118 LSD1 inhibitor III CBB1007 SB203580 Trichostatin A119 LSD1 inhibitor I SB203580 Trichostatin A 120 TranylcypromineSB203580 Trichostatin A 121 LSD1 inhibitor IV RN SB203580 istodax 122LSD1 inhibitor II S2101 SB203580 istodax 123 LSD1 inhibitor LSD1-C76SB203580 istodax 124 LSD1 inhibitor II CBB1007 SB203580 istodax 125 LSD1inhibitor I SB203580 istodax 126 Tranylcypromine SB203580 istodax 127LSD1 inhibitor IV RN CHIR99021 Trichostatin A 128 LSD1 inhibitor IIS2101 CHIR99021 Trichostatin A 129 LSD1 inhibitor LSD1-C76 CHIR99021Trichostatin A 130 LSD1 inhibitor III CBB1007 CHIR99021 Trichostatin A131 LSD1 inhibitor I CHIR99021 Trichostatin A 132 TranylcypromineCHIR99021 Trichostatin A 133 LSD1 inhibitor IV RN CHIR99021 istodax 134LSD1 inhibitor II S2101 CHIR99021 istodax 135 LSD1 inhibitor LSD1-C76CHIR99021 istodax 136 LSD1 inhibitor III CBB1007 CHIR99021 istodax 137LSD1 inhibitor I CHIR99021 istodax 138 Tranylcypromine CHIR99021 istodax139 ALK5 inhibitor II CHIR99021 Trichostatin A 140 LY364947 CHIR99021Trichostatin A 141 A83-01 CHIR99021 Trichostatin A 142 DMH1 CHIR99021Trichostatin A 143 ALK5 inhibitor II CHIR99021 istodax 144 LY364947CHIR99021 istodax 145 A83-01 CHIR99021 istodax 146 DMH1 CHIR99021istodax 147 SB203580 CHIR99021 Trichostatin A 148 SB203580 CHIR99021istodax

TABLE 9 No. 4-Component Combination 1 LSD1 inhibitor IV RN ALK5inhibitor II SB203580 CHIR99021 2 LSD1 inhibitor IV RN LY364947 SB203580CHIR99021 3 LSD1 inhibitor IV RN A83-01 SB203580 CHIR99021 4 LSD1inhibitor IV RN DMH1 SB203580 CHIR99021 5 LSD1 inhibitor II S2101 ALK5inhibitor II SB203580 CHIR99021 6 LSD1 inhibitor II S2101 LY364947SB203580 CHIR99021 7 LSD1 inhibitor II S2101 A83-01 SB203580 CHIR99021 8LSD1 inhibitor II S2101 DMH1 SB203580 CHIR99021 9 LSD1 inhibitorLSD1-C76 ALK5 inhibitor II SB203580 CHIR99021 10 LSD1 inhibitor LSD1-C76LY364947 SB203580 CHIR99021 11 LSD1 inhibitor LSD1-C76 A83-01 SB203580CHIR99021 12 LSD1 inhibitor LSD1-C76 DMH1 SB203580 CHIR99021 13 LSD1inhibitor III CBB1007 ALK5 inhibitor II SB203580 CHIR99021 14 LSD1inhibitor III CBB1007 LY364947 SB203580 CHIR99021 15 LSD1 inhibitor IIICBB1007 A83-01 SB203580 CHIR99021 16 LSD1 inhibitor III CBB1007 DMH1SB203580 CHIR99021 17 LSD1 inhibitor I ALK5 inhibitor II SB203580CHIR99021 18 LSD1 inhibitor I LY364947 SB203580 CHIR99021 19 LSD1inhibitor I A83-01 SB203580 CHIR99021 20 LSD1 inhibitor I DMH1 SB203580CHIR99021 21 Tranylcypromine ALK5 inhibitor II SB203580 CHIR99021 22Tranylcypromine LY364947 SB203580 CHIR99021 23 Tranylcypromine A83-01SB203580 CHIR99021 24 Tranylcypromine DMH1 SB203580 CHIR99021 25 LSD1inhibitor IV RN ALK5 inhibitor II SB203580 Trichostatin A 26 LSD1inhibitor IV RN LY364947 SB203580 Trichostatin A 27 LSD1 inhibitor IV RNA83-01 SB203580 Trichostatin A 28 LSD1 inhibitor IV RN DMH1 SB203580Trichostatin A 29 LSD1 inhibitor II S2101 ALK5 inhibitor II SB203580Trichostatin A 30 LSD1 inhibitor II S2101 LY364947 SB203580 TrichostatinA 31 LSD1 inhibitor II S2101 A83-01 SB203580 Trichostatin A 32 LSD1inhibitor II S2101 DMH1 SB203580 Trichostatin A 33 LSD1 inhibitorLSD1-C76 ALK5 inhibitor II SB203580 Trichostatin A 34 LSD1 inhibitorLSD1-C76 LY364947 SB203580 Trichostatin A 35 LSD1 inhibitor LSD1-C76A83-01 SB203580 Trichostatin A 36 LSD1 inhibitor LSD1-C76 DMH1 SB203580Trichostatin A 37 LSD1 inhibitor III CBB1007 ALK5 inhibitor II SB203580Trichostatin A 38 LSD1 inhibitor III CBB1007 LY364947 SB203580Trichostatin A 39 LSD1 inhibitor III CBB1007 A83-01 SB203580Trichostatin A 40 LSD1 inhibitor III CBB1007 DMH1 SB203580 TrichostatinA 41 LSD1 inhibitor I ALK5 inhibitor II SB203580 Trichostatin A 42 LSD1inhibitor I LY364947 SB203580 Trichostatin A 43 LSD1 inhibitor I A83-01SB203580 Trichostatin A 44 LSD1 inhibitor I DMH1 SB203580 Trichostatin A45 Tranylcypromine ALK5 inhibitor II SB203580 Trichostatin A 46Tranylcypromine LY364947 SB203580 Trichostatin A 47 TranylcypromineA83-01 SB203580 Trichostatin A 48 Tranylcypromine DMH1 SB203580Trichostatin A 49 LSD1 inhibitor IV RN ALK5 inhibitor II SB203580istodax 50 LSD1 inhibitor IV RN LY364947 SB203580 istodax 51 LSD1inhibitor IV RN A83-01 SB203580 istodax 52 LSD1 inhibitor IV RN DMH1SB203580 istodax 53 LSD1 inhibitor II S2101 ALK5 inhibitor II SB203580istodax 54 LSD1 inhibitor II S2101 LY364947 SB203580 istodax 55 LSD1inhibitor II S2101 A83-01 SB203580 istodax 56 LSD1 inhibitor II S2101DMH1 SB203580 istodax 57 LSD1 inhibitor LSD1-C76 ALK5 inhibitor IISB203580 istodax 58 LSD1 inhibitor LSD1-C76 LY364947 SB203580 istodax 59LSD1 inhibitor LSD1-C76 A83-01 SB203580 istodax 60 LSD1 inhibitorLSD1-C76 DMH1 SB203580 istodax 61 LSD1 inhibitor III CBB1007 ALK5inhibitor II SB203580 istodax 62 LSD1 inhibitor III CBB1007 LY364947SB203580 istodax 63 LSD1 inhibitor III CBB1007 A83-01 SB203580 istodax64 LSD1 inhibitor III CBB1007 DMH1 SB203580 istodax 65 LSD1 inhibitor IALK5 inhibitor II SB203580 istodax 66 LSD1 inhibitor I LY364947 SB203580istodax 67 LSD1 inhibitor I A83-01 SB203580 istodax 68 LSD1 inhibitor IDMH1 SB203580 istodax 66 Tranylcypromine ALK5 inhibitor II SB203580istodax 70 Tranylcypromine LY364947 SB203580 istodax 71 TranylcypromineA83-01 SB203580 istodax 72 Tranylcypromine DMH1 SB203580 istodax 73 ALK5inhibitor II SB203580 CHIR99021 Trichostatin A 74 LY364947 SB203580CHIR99021 Trichostatin A 75 A83-01 SB203580 CHIR99021 Trichostatin A 76DMH1 SB203580 CHIR99021 Trichostatin A 77 ALK5 inhibitor II SB203580CHIR99021 istodax 78 LY364947 SB203580 CHIR99021 istodax 79 A83-01SB203580 CHIR99021 istodax 80 DMH1 SB203580 CHIR99021 istodax 81 LSD1inhibitor IV RN SB203580 CHIR99021 Trichostatin A 82 LSD1 inhibitor IIS2101 SB203580 CHIR99021 Trichostatin A 83 LSD1 inhibitor LSD1-C76SB203580 CHIR99021 Trichostatin A 84 LSD1 inhibitor III CBB1007 SB203580CHIR99021 Trichostatin A 85 LSD1 inhibitor I SB203580 CHIR99021Trichostatin A 86 Tranylcypromine SB203580 CHIR99021 Trichostatin A 87LSD1 inhibitor IV RN SB203580 CHIR99021 istodax 88 LSD1 inhibitor IIS2101 SB203580 CHIR99021 istodax 89 LSD1 inhibitor LSD1-C76 SB203580CHIR99021 istodax 90 LSD1 inhibitor III CBB1007 SB203580 CHIR99021istodax 91 LSD1 inhibitor I SB203580 CHIR99021 istodax 92Tranylcypromine SB203580 CHIR99021 istodax 93 LSD1 inhibitor IV RN ALK5inhibitor II CHIR99021 Trichostatin A 94 LSD1 inhibitor IV RN LY364947CHIR99021 Trichostatin A 95 LSD1 inhibitor IV RN A83-01 CHIR99021Trichostatin A 96 LSD1 inhibitor IV RN DMH1 CHIR99021 Trichostatin A 97LSD1 inhibitor II S2101 ALK5 inhibitor II CHIR99021 Trichostatin A 98LSD1 inhibitor II S2101 LY364947 CHIR99021 Trichostatin A 99 LSD1inhibitor II S2101 A83-01 CHIR99021 Trichostatin A 100 LSD1 inhibitor IIS2101 DMH1 CHIR99021 Trichostatin A 101 LSD1 inhibitor LSD1-C76 ALK5inhibitor II CHIR99021 Trichostatin A 102 LSD1 inhibitor LSD1-C76LY364947 CHIR99021 Trichostatin A 103 LSD1 inhibitor LSD1-C76 A83-01CHIR99021 Trichostatin A 104 LSD1 inhibitor LSD1-C76 DMH1 CHIR99021Trichostatin A 105 LSD1 inhibitor III CBB1007 ALK5 inhibitor IICHIR99021 Trichostatin A 106 LSD1 inhibitor III CBB1007 LY364947CHIR99021 Trichostatin A 107 LSD1 inhibitor III CBB1007 A83-01 CHIR99021Trichostatin A 108 LSD1 inhibitor III CBB1007 DMH1 CHIR99021Trichostatin A 109 LSD1 inhibitor I ALK5 inhibitor II CHIR99021Trichostatin A 110 LSD1 inhibitor I LY364947 CHIR99021 Trichostatin A111 LSD1 inhibitor I A83-01 CHIR99021 Trichostatin A 112 LSD1 inhibitorI DMH1 CHIR99021 Trichostatin A 113 Tranylcypromine ALK5 inhibitor IICHIR99021 Trichostatin A 114 Tranylcypromine LY364947 CHIR99021Trichostatin A 115 Tranylcypromine A83-01 CHIR99021 Trichostatin A 116Tranylcypromine DMH1 CHIR99021 Trichostatin A 117 LSD1 inhibitor IV RNALK5 inhibitor II CHIR99021 istodax 118 LSD1 inhibitor IV RN LY364947CHIR99021 istodax 119 LSD1 inhibitor IV RN A83-01 CHIR99021 istodax 120LSD1 inhibitor IV RN DMH1 CHIR99021 istodax 121 LSD1 inhibitor II S2101ALK5 inhibitor II CHIR99021 istodax 122 LSD1 inhibitor II S2101 LY364947CHIR99021 istodax 123 LSD1 inhibitor II S2101 A83-01 CHIR99021 istodax124 LSD1 inhibitor II S2101 DMH1 CHIR99021 istodax 125 LSD1 inhibitorLSD1-C76 ALK5 inhibitor II CHIR99021 istodax 126 LSD1 inhibitor LSD1-C76LY364947 CHIR99021 istodax 127 LSD1 inhibitor LSD1-C76 A83-01 CHIR99021istodax 128 LSD1 inhibitor LSD1-C76 DMH1 CHIR99021 istodax 129 LSD1inhibitor III CBB1007 ALK5 inhibitor II CHIR99021 istodax 130 LSD1inhibitor III CBB1007 LY364947 CHIR99021 istodax 131 LSD1 inhibitor IIICBB1007 A83-01 CHIR99021 istodax 132 LSD1 inhibitor III CBB1007 DMH1CHIR99021 istodax 133 LSD1 inhibitor I ALK5 inhibitor II CHIR99021istodax 134 LSD1 inhibitor I LY364947 CHIR99021 istodax 135 LSD1inhibitor I A83-01 CHIR99021 istodax 136 LSD1 inhibitor I DMH1 CHIR99021istodax 137 Tranylcypromine ALK5 inhibitor II CHIR99021 istodax 138Tranylcypromine LY364947 CHIR99021 istodax 139 Tranylcypromine A83-01CHIR99021 istodax 140 Tranylcypromine DMH1 CHIR99021 istodax

TABLE 10 No. 5-Component Combination 1 LSD1 inhibitor IV RN ALK5inhibitor II SB203580 CHIR99021 Trichostatin A 2 LSD1 inhibitor IV RNLY364947 SB203580 CHIR99021 Trichostatin A 3 LSD1 inhibitor IV RN A83-01SB203580 CHIR99021 Trichostatin A 4 LSD1 inhibitor IV RN DMH1 SB203580CHIR99021 Trichostatin A 5 LSD1 inhibitor II S2101 ALK5 inhibitor IISB203580 CHIR99021 Trichostatin A 6 LSD1 inhibitor II S2101 LY364947SB203580 CHIR99021 Trichostatin A 7 LSD1 inhibitor II S2101 A83-01SB203580 CHIR99021 Trichostatin A 8 LSD1 inhibitor II S2101 DMH1SB203580 CHIR99021 Trichostatin A 9 LSD1 inhibitor LSD1-C76 ALK5inhibitor II SB203580 CHIR99021 Trichostatin A 10 LSD1 inhibitorLSD1-C76 LY364947 SB203580 CHIR99021 Trichostatin A 11 LSD1 inhibitorLSD1-C76 A83-01 SB203580 CHIR99021 Trichostatin A 12 LSD1 inhibitorLSD1-C76 DMH1 SB203580 CHIR99021 Trichostatin A 13 LSD1 inhibitor IIICBB1007 ALK5 inhibitor II SB203580 CHIR99021 Trichostatin A 14 LSD1inhibitor III CBB1007 LY364947 SB203580 CHIR99021 Trichostatin A 15 LSD1inhibitor III CBB1007 A83-01 SB203580 CHIRP9021 Trichostatin A 16 LSD1inhibitor III CBB1007 DMH1 SB203580 CHIR99021 Trichostatin A 17 LSD1inhibitor I ALK5 inhibitor II SB203580 CHIR99021 Trichostatin A 18 LSD1inhibitor I LY364947 SB203580 CHIR99021 Trichostatin A 19 LSD1 inhibitorI A83-01 SB203580 CHIR99021 Trichostatin A 20 LSD1 inhibitor I DMH1SB203580 CHIR99021 Trichostatin A 21 Tranylcypromine ALK5 inhibitor IISB203580 CHIR99021 Trichostatin A 22 Tranylcypromine LY364947 SB203580CHIR99021 Trichostatin A 23 Tranylcypromine A83-01 SB203580 CHIR99021Trichostatin A 24 Tranylcypromine DMH1 SB203580 CHIR99021 Trichostatin A25 LSD1 inhibitor IV RN ALK5 inhibitor II SB203580 CHIR99021 istodax 26LSD1 inhibitor IV RN LY364947 SB203580 CHIR99021 istodax 27 LSD1inhibitor IV RN A83-01 SB203580 CHIR99021 istodax 28 LSD1 inhibitor IVRN DMH1 SB203580 CHIR99021 istodax 29 LSD1 inhibitor II S2101 ALK5inhibitor II SB203580 CHIR99021 istodax 30 LSD1 inhibitor II S2101LY364947 SB203580 CHIR99021 istodax 31 LSD1 inhibitor II S2101 A83-01SB203580 CHIR99021 istodax 32 LSD1 inhibitor II S2101 DMH1 SB203580CHIR99021 istodax 33 LSD1 inhibitor LSD1-C76 ALK5 inhibitor II SB203580CHIR99021 istodax 34 LSD1 inhibitor LSD1-C76 LY364947 SB203580 CHIR99021istodax 35 LSD1 inhibitor LSD1-C76 A83-01 SB203580 CHIR99021 istodax 36LSD1 inhibitor LSD1-C76 DMH1 SB203580 CHIR99021 istodax 37 LSD1inhibitor III CBB1007 ALK5 inhibitor II SB203580 CHIR99021 istodax 38LSD1 inhibitor III CBB1007 LY364947 SB203580 CHIR99021 istodax 39 LSD1inhibitor III CBB1007 A83-01 SB203580 CHIR99021 istodax 40 LSD1inhibitor III CBB1007 DMH1 SB203580 CHIR99021 istodax 41 LSD1 inhibitorI ALK5 inhibitor II SB203580 CHIR99021 istodax 42 LSD1 inhibitor ILY364947 SB203580 CHIR99021 istodax 43 LSD1 inhibitor I A83-01 SB203580CHIR99021 istodax 44 LSD1 inhibitor I DMH1 SB203580 CHIR99021 istodax 45Tranylcypromine ALK5 inhibitor II SB203580 CHIR99021 istodax 46Tranylcypromine LY364947 SB203580 CHIR99021 istodax 47 TranylcypromineA83-01 SB203580 CHIR99021 istodax 48 Tranylcypromine DMH1 SB203580CHIR99021 istodaxAdditional Agents that can be Used to Induce Expansion, Enrichment, andMaintenance of Hematopoietic Stem Cells During Ex Vivo Culturing

Other compounds may additionally be used in conjunction with thecompositions and methods of the present invention in order to expand,enrich, and/or maintain hematopoietic stem cells during ex vivoculturing. Examples of these compounds include antagonists of the arylhydrocarbon receptor (AHR), such as StemRegenin 1 (SR1), a smallmolecule that promotes expansion and self-renewal of human CD34+peripheral blood and cord blood hematopoietic stem cells. SR1 has beendescribed, e.g., in US 2014/0369973; Boitano et al. Science 1345 (2010);and Smith et al. Journal of Pharmacology and Experimental Therapeutics338:318 (2011), the disclosures of each of which are incorporated hereinby reference. Other AHR inhibitors that may be used in conjunction withthe compositions and methods of the invention include SR1 analogs, suchas those that contain various aryl and aliphatic substituents about the6-aminopurine core (e.g., those described in US 2014/0369973, thedisclosure of which is incorporated herein by reference). Additionalexamples of AHR antagonists include the stilbene derivatives(E)-1-(4′-trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)-ethene,(E)-1-(4′-methoxyphenyl)-2-(3,5-dichlorophenyl)-ethene, and(E)-1-(4′-chlorophenyl)-2-(3,5-dichlorophenyl)-ethene, as described in,e.g., WO 2004/041758, the disclosure of which is incorporated herein byreference. Additional stilbene derivatives useful for the inhibition ofthe AHR include 3,5,4′-trihydroxystilbenes (e.g., resveratrols and, inparticular, trans-resveratrol); 3,4,3′,5-tetrahydroxystilbene (alsoreferred to as piceatannol); 2,3′,4′,5′-tetrahydrostilbene (alsoreferred to as oxyresveratrol); as well as 4,4′-dihydroxystilbenes andglycosides (e.g., galactosides, lactosides, mannosides, piceosides, andfructosides thereof) as described in, e.g., WO 1999/056737, thedisclosure of which is incorporated herein by reference. Anotherexemplary AHR antagonist is 2-methyl-2H-pyrazole-3-carboxylicacid-(2-methyl-4-o-tolylazophenyl)-amide (also referred to as CH-223191,described in detail in, e.g., Kim et al. Molecular Pharmacology 69:1871(2006); as well as in WO 2009/115807; the disclosures of each of whichare incorporated herein by reference).

Additional agents that can be used in conjunction with the methods ofthe invention include UM171, another small molecule that has been shownto induce hematopoietic stem cell expansion. UM171 is described, e.g.,in Fares, et al., Science, 345(6203):1509-1512 (2014), the disclosure ofwhich is incorporated herein by reference. Other agents that can be usedwith the compositions and methods of the invention include UM171analogs, such as those described in WO 2013/110198, the disclosure ofwhich is incorporated herein by reference. Particularly useful analogsof UM171 that can be used in conjunction with the compositions andmethods of the invention include compound Nos. 1-55 recited in WO2013/110198 and the compounds disclosed in Table 11 herein. A furtherexample of a class of compound that can additionally be used inconjunction with the compositions and methods of the present inventionincludes prostaglandins. Particularly useful prostaglandins includeprostaglandin dmPGE2, described, e.g., in U.S. Pat. Nos. 8,551,782 and8,168,428, the disclosures of each of which are incorporated herein byreference. Additional compounds that can be used in conjunction with thecompositions and methods described herein include inhibitors of theSirtuin 1 (SIRT1) protein, such as nicotinamide (described, e.g., inPeled et al. Experimental Hematology 40:342 (2012)) and cambinol(described, e.g., in Lugrin et al. Biochimica Biophysica Acta 1833:1498(2013)), the disclosures of each of which are incorporated herein byreference.

Additional agents that can be contacted with hematopoietic stem cells incombination with one or more agents that together exhibit two or moreactivities selected from the group consisting of modulation of histonemethylation, inhibition of TGFβ signaling, inhibition of p38 signaling,activation of canonical Wnt signaling, and modulation of histoneacetylation include activators of the Notch signal transduction pathway.Agonists of Notch signaling include but are not limited to proteins thatcontain portions of toporythmic proteins such as Delta, Serrate orJagged (see, e.g., Lindsell et al., Cell 80: 909-917 (1995), thedisclosure of which is incorporated herein by reference) that mediatebinding to Notch and/or mediate Notch activity, and nucleic acidsencoding the foregoing, as well as proteins, nucleic acids, smallmolecules, or derivatives thereof that regulate activity or geneexpression of these proteins. Notch signaling agonists also include aprotein or derivative or fragment thereof comprising a functionallyactive fragment such as a fragment of a Notch ligand that mediatesbinding to a Notch protein.

Notch activity is promoted by the binding of Notch ligands (e.g., Deltaligands and Serrate ligands) to the extracellular portion of the Notchreceptor. Endogenous Notch ligands are typically membrane-bound onadjacent cells. As such, Notch ligands for use with the compositions andmethods of the invention may be incubated with hematopoietic stem cellsin solution as soluble protein factors or immobilized on a solid surface(e.g., a tissue culture plate, bead, or nanomatrix). For example, fulllength Notch ligands expressed on the surface of a cell induces theactivation of the Notch signaling cascade in a neighboring cell uponcontact of the ligand with the Notch receptor. Notch signaling agonistsfor use with the compositions and methods of the invention thus includesoluble, optionally truncated Delta or Serrate (e.g., Jagged) moleculesthat contain the extracellular domains or Notch-binding portions, aswell as forms of these proteins immobilized on a surface, such as thesolid surface of a tissue culture plate, water-miscible bead, ornanomatrix. Such soluble proteins can be immobilized on a solid surfaceby an antibody or interacting protein, for example an antibody directedto an epitope tag with which a Delta or a Serrate is expressed as afusion protein (e.g., a myc epitope tag, which is recognized by theantibody 9E10) or a protein which interacts with an epitope tag withwhich a Delta or a Serrate is expressed as a fusion protein (e.g., animmunoglobulin epitope tag, which is bound by Protein A) as described inUS 2014/0369973, the disclosure of which is incorporated herein byreference. Exemplary agonists of Notch signaling additionally include anotch polypeptide, deltex polypeptide, mastermind polypeptide, splitpolypeptide, hairless polypeptide, RBP-Jκ polypeptide, or heslpolypeptide as described in US 2011/0091448, the disclosure of which isincorporated herein by reference.

Agents such as those described above (for example, an AHR antagonist,such as SR1, optionally in combination with UM171, dmPGE2, a Notchsignaling agonist, and/or a SIRT1 inhibitor, such as nicotinamide orcambinol) may be included as additional compounds that contacthematopoietic stem cells and that are in contact with one or more agentsthat together exhibit two or more activities selected from the groupconsisting of modulation of histone methylation, inhibition of TGFβsignaling, inhibition of p38 signaling, activation of canonical Wntsignaling, and modulation of histone acetylation. For instance,hematopoietic stem cells in contact with one or more of these agents mayadditionally be contacted with an AHR antagonist, UM171, dmPGE2, a Notchsignaling agonist, and/or a SIRT1 inhibitor, such as nicotinamide orcambinol, according to distinct incubation regimens, such that one ormore of these compounds are introduced to hematopoietic stem cells atvarious times during a culture period. Alternatively, these agents maybe incubated with hematopoietic stem cells simultaneously when desired.

Hematopoietic Stem Cell Mobilization

Hematopoietic stem cells for use with the compositions and methods ofthe invention may arise from a variety of cell types. For instance,hematopoietic cells for methods of expansion, enrichment, andmaintenance of hematopoietic stem cell functional potential as recitedherein may are derived from mononuclear cells prior to the treatment ofthese cells with one or more agents that together exhibit two or moreactivities selected from the group consisting of modulation of histonemethylation, inhibition of TGFβ signaling, inhibition of p38 signaling,activation of canonical Wnt signaling, and modulation of histoneacetylation. Human hematopoietic stem cells may optionally be CD34+cells prior to the treatment with one or more of these agents. Forinstance, human hematopoietic stem cells may be within populations withcell surface phenotypes including CD34+, CD34+CD38-, CD34+CD38-CD90+,CD34+CD38-CD90+CD45RA−, or CD34+CD38-CD90+CD45RA-CD49F+ cells prior tothe treatment with one or more of these agents.

Hematopoietic stem cells may additionally are derived from human bonemarrow. Alternatively, hematopoietic stem cells may be derived fromhuman cord blood or mobilized peripheral blood. Hematopoietic stem cellsobtained from human peripheral blood may be mobilized by one of avariety of strategies. Exemplary agents that can be used to inducemobilization of hematopoietic stem cells from the bone marrow intoperipheral blood include chemokine (C—X—C motif) receptor 4 (CXCR4)antagonists, such as AMD3100 (also known as Plerixafor and MOZOBIL™(Genzyme, Boston, Mass.)) and granulocyte colony-stimulating factor(GCSF), the combination of which has been shown to rapidly mobilizeCD34⁺ cells in clinical experiments. Additionally, chemokine (C—X—Cmotif) ligand 2 (CXCL2, also referred to as GROβ) represents anotheragent capable of inducing hematopoietic stem cell mobilization to frombone marrow to peripheral blood. Agents capable of inducing mobilizationof hematopoietic stem cells for use with the compositions and methods ofthe invention may be used in combination with one another. For instance,CXCR4 antagonists (e.g., AMD3100), CXCL2, and/or GCSF may beadministered to a subject sequentially or simultaneously in a singlemixture in order to induce mobilization of hematopoietic stem cells frombone marrow into peripheral blood. The use of these agents as inducersof hematopoietic stem cell mobilization is described, e.g., in Pelus,Current Opinion in Hematology 15:285 (2008), the disclosure of which isincorporated herein by reference.

Modulating Target Gene Expression in Hematopoietic Stem Cells

The compositions and methods of the invention further provide strategiesfor regulating the expression of target genes in populations ofhematopoietic stem cells. For instance, a population of hematopoieticstem cells may be expanded, enriched or maintained ex vivo according tothe methods of the invention and may additionally be geneticallymodified so as to exhibit an altered gene expression pattern.Alternatively, a population of cells may be enriched with hematopoieticstem cells, or a population of hematopoietic stem cells may bemaintained in a multi-potent state, and the cells may further bemodified using established genome editing techniques known in the art.For instance, one may use a genome editing procedure to promote theexpression of an exogenous gene or inhibit the expression of anendogenous gene within a hematopoietic stem cell. Importantly,populations of hematopoietic stem cells may be expanded, enriched, ormaintained in a multi-potent state according to the methods of theinvention recited herein and subsequently genetically modified so as toexpress a desired target gene, or populations of these cells may begenetically modified first and then expanded, enriched, or maintained ina multi-potent state. A wide array of methods has been established forthe incorporation of target genes into the genome of a cell (e.g., amammalian cell, such as a murine or human cell) so as to facilitate theexpression of such genes.

Polynucleotides Encoding Target Genes

One example of a platform that can be used to facilitate the expressionof a target gene in a hematopoietic stem cell is by the integration ofthe polynucleotide encoding a target gene into the nuclear genome of thecell. A variety of techniques have been developed for the introductionof exogenous genes into a eukaryotic genome. One such technique involvesthe insertion of a target gene into a vector, such as a viral vector.Vectors for use with the compositions and methods of the invention canbe introduced into a cell by a variety of methods, includingtransformation, transfection, direct uptake, projectile bombardment, andby encapsulation of the vector in a liposome. Examples of suitablemethods of transfecting or transforming cells include calcium phosphateprecipitation, electroporation, microinjection, infection, lipofectionand direct uptake. Such methods are, described in more detail, forexample, in Green, et al., Molecular Cloning: A Laboratory Manual,Fourth Edition, Cold Spring Harbor University Press, New York (2014);and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York (2015), the disclosures of each of which areincorporated herein by reference.

Exogenous genes can also be introduced into a mammalian cell through theuse of a vector containing the gene of interest to cell membranephospholipids. For example, vectors can be targeted to the phospholipidson the extracellular surface of the cell membrane by linking the vectormolecule to a VSV-G protein, a viral protein with affinity for all cellmembrane phospholipids. Viral vectors containing the VSV-G protein aredescribed in further detail, e.g., in U.S. Pat. No. 5,512,421; and inU.S. Pat. No. 5,670,354, the disclosures of each of which areincorporated by reference herein.

Recognition and binding of the polynucleotide encoding a target gene bymammalian RNA polymerase is an important molecular event for geneexpression to occur. As such, one may include sequence elements withinthe polynucleotide that exhibit a high affinity for transcriptionfactors that recruit RNA polymerase and promote the assembly of thetranscription complex at the transcription initiation site. Suchsequence elements include, e.g., a mammalian promoter, the sequence ofwhich can be recognized and bound by specific transcription initiationfactors and ultimately RNA polymerase. Alternatively, promoters derivedfrom viral genomes can be used for the stable expression of target genesin mammalian cells. Examples of functional viral promoters that can beused to promote mammalian expression of these enzymes include adenoviruslate promoter, vaccinia virus 7.5K promoter, SV40 promoter,cytomegalovirus promoter, mouse mammary tumor virus (MMTV) promoter, LTRpromoter of HIV, promoter of moloney virus, Epstein barr virus (EBV)promoter, Rous sarcoma virus (RSV) promoter, and the cytomegalovirus(CMV) promoter. Additional viral promoters include the SV40 latepromoter from simian virus 40, the Baculovirus polyhedronenhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSVtk) promoter, and the 35S promoter from Cauliflower Mosaic Virus.Suitable phage promoters for use with the compositions and methods ofthe invention include, but are not limited to, the E. coli T7 and T3phage promoters, the S. typhimurium phage SP6 promoter, B. subtilis SP01phage and B. subtilis phage phi 29 promoters, and N4 phage and K11 phagepromoters as described in U.S. Pat. No. 5,547,892, the disclosure ofwhich is incorporated herein by reference.

Upon incorporation of a polynucleotide encoding a target gene has beenincorporated into the genome of a cell (e.g., the nuclear genome of ahematopoietic stem cell), the transcription of this polynucleotide canbe induced by methods known in the art. For example expression can beinduced by exposing the mammalian cell to an external chemical reagent,such as an agent that modulates the binding of a transcription factorand/or RNA polymerase to the mammalian promoter and thus regulate geneexpression. The chemical reagent can serve to facilitate the binding ofRNA polymerase and/or transcription factors to the mammalian promoter,e.g., by removing a repressor protein that has bound the promoter.Alternatively, the chemical reagent can serve to enhance the affinity ofthe mammalian promoter for RNA polymerase and/or transcription factorssuch that the rate of transcription of the gene located downstream ofthe promoter is increased in the presence of the chemical reagent.Examples of chemical reagents that potentiate polynucleotidetranscription by the above mechanisms include tetracycline anddoxycycline. These reagents are commercially available (LifeTechnologies, Carlsbad, Calif.) and can be administered to a mammaliancell in order to promote gene expression according to establishedprotocols.

Other DNA sequence elements that may be included in polynucleotides foruse with the compositions and methods of the invention include enhancersequences. Enhancers represent another class of regulatory elements thatinduce a conformational change in the polynucleotide comprising the geneof interest such that the DNA adopts a three-dimensional orientationthat is favorable for binding of transcription factors and RNApolymerase at the transcription initiation site. Thus, polynucleotidesfor use with the compositions and methods of the invention include thosethat encode a target gene and additionally include a mammalian enhancersequence. Many enhancer sequences are now known from mammalian genes,and examples include enhancers from the genes that encode mammalianglobin, elastase, albumin, α-fetoprotein, and insulin. Enhancers for usewith the compositions and methods of the invention also include thosethat are derived from the genetic material of a virus capable ofinfecting a eukaryotic cell. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100-270), the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. Additional enhancersequences that induce activation of eukaryotic gene transcription aredisclosed in Yaniv, et al., Nature, 297:17-18 (1982), the disclosure ofwhich is incorporated herein by reference. An enhancer may be splicedinto a vector containing a polynucleotide encoding a target gene, forexample, at a position 5′ or 3′ to this gene. In a preferredorientation, the enhancer is positioned at the 5′ side of the promoter,which in turn is located 5′ relative to the polynucleotide encoding thetarget gene.

In addition to promoting high rates of transcription and translation,stable expression of an exogenous gene in a hematopoietic stem cell canbe achieved by integration of the polynucleotide comprising the geneinto the nuclear DNA of the cell. A variety of vectors for the deliveryand integration of polynucleotides encoding exogenous proteins into thenuclear DNA of a mammalian cell have been developed. Examples ofexpression vectors are disclosed in, e.g., WO 1994/11026, the disclosureof which is incorporated herein by reference. Expression vectors for usewith the compositions and methods of the invention contain apolynucleotide sequence that encodes a target gene, as well as, e.g.,additional sequence elements used for the expression of these enzymesand/or the integration of these polynucleotide sequences into the genomeof a mammalian cell. Certain vectors that can be used for the expressionof target genes include plasmids that contain regulatory sequences, suchas promoter and enhancer regions, which direct gene transcription. Otheruseful vectors for expression of target genes contain polynucleotidesequences that enhance the rate of translation of these genes or improvethe stability or nuclear export of the mRNA that results from genetranscription. These sequence elements often encode features within RNAtranscripts that enhance the nuclear export, cytosolic half-life, andribosomal affinity of these molecules, e.g., 5′ and 3′ untranslatedregions, an internal ribosomal entry site (IRES), and polyadenylationsignal site in order to direct efficient transcription of the genecarried on the expression vector. Exemplary expression vectors may alsocontain a polynucleotide encoding a marker for selection of cells thatcontain such a vector. Non-limiting examples of a suitable markerinclude genes that encode resistance to antibiotics, such as ampicillin,chloramphenicol, kanamycin, or nourseothricin.

Vectors for the Expression of Target Genes

Viral genomes provide a rich source of vectors that can be used for theefficient delivery of exogenous genes into a mammalian cell. Viralgenomes are particularly useful vectors for gene delivery because thepolynucleotides contained within such genomes are typically incorporatedinto the nuclear genome of a mammalian cell by generalized orspecialized transduction. These processes occur as part of the naturalviral replication cycle, and often do not require added proteins orreagents in order to induce gene integration. Examples of viral vectorsinclude a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, andAd48), parvovirus (e.g., adeno-associated viruses), coronavirus,negative strand RNA viruses such as orthomyxovirus (e.g., influenzavirus), rhabdovirus (e.g., rabies and vesicular stomatitis virus),paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses,such as picornavirus and alphavirus, and double stranded DNA virusesincluding herpes virus (e.g., Herpes Simplex virus types 1 and 2,Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia,modified vaccinia Ankara (MVA), fowlpox and canarypox). Other virusesinclude Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus,hepadnavirus, and hepatitis virus, for example. Examples of retrovirusesinclude: avian leukosis-sarcoma, mammalian C-type, B-type viruses,D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.,Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996, the disclosure of which is incorporatedherein by reference). Other examples of viral vectors include murineleukemia viruses, murine sarcoma viruses, mouse mammary tumor virus,bovine leukemia virus, feline leukemia virus, feline sarcoma virus,avian leukemia virus, human T-cell leukemia virus, baboon endogenousvirus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simianimmunodeficiency virus, simian sarcoma virus, Rous sarcoma virus andlentiviruses. Other examples of vectors are described in, e.g., U.S.Pat. No. 5,801,030, the disclosure of which is incorporated herein byreference.

Additional Transfection Methods

Other techniques that can be used to introduce a polynucleotide, such asDNA or RNA (e.g., mRNA, tRNA, siRNA, miRNA, shRNA, chemically modifiedRNA) into a mammalian cell are well known in the art. For instance,electroporation can be used to permeabilize mammalian cells by theapplication of an electrostatic potential. Mammalian cells, such ashematopoietic stem cells, subjected to an external electric field inthis manner are subsequently predisposed to the uptake of exogenousnucleic acids. Electroporation of mammalian cells is described indetail, e.g., in Chu et al. Nucleic Acids Research 15:1311 (1987), thedisclosure of which is incorporated herein by reference. A similartechnique, Nucleofection™, utilizes an applied electric field in orderto stimulate the update of exogenous polynucleotides into the nucleus ofa eukaryotic cell. Nucleofection™ and protocols useful for performingthis technique are described in detail, e.g., in Distler et al.Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114,the disclosures of each of which are incorporated herein by reference.

Additional techniques useful for the transfection of hematopoietic stemcells include the squeeze-poration methodology. This technique inducesthe rapid mechanical deformation of cells in order to stimulate theuptake of exogenous DNA through membranous pores that form in responseto the applied stress. This technology is advantageous in that a vectoris not required for delivery of nucleic acids into a cell, such as ahematopoietic stem cell. Squeeze-poration is described in detail, e.g.,in Sharei et al. Journal of Visualized Experiments 81:e50980 (2013), thedisclosure of which is incorporated herein by reference.

Lipofection represents another technique useful for transfection ofhematopoietic stem cells. This method involves the loading of nucleicacids into a liposome, which often presents cationic functional groups,such as quaternary or protonated amines, towards the liposome exterior.This promotes electrostatic interactions between the liposome and a celldue to the anionic nature of the cell membrane, which ultimately leadsto uptake of the exogenous nucleic acids, e.g., by direct fusion of theliposome with the cell membrane or by endocytosis of the complex.Lipofection is described in detail, e.g., in U.S. Pat. No. 7,442,386,the disclosure of which is incorporated herein by reference. Similartechniques that exploit ionic interactions with the cell membrane toprovoke the uptake of foreign nucleic acids include contacting a cellwith a cationic polymer-nucleic acid complex. Exemplary cationicmolecules that associate with polynucleotides so as to impart a positivecharge favorable for interaction with the cell membrane includeactivated dendrimers (described, e.g., in Dennig, Topics in CurrentChemistry 228:227 (2003), the disclosure of which is incorporated hereinby reference) and diethylaminoethyl (DEAE)-dextran, the use of which asa transfection agent is described in detail, e.g., in Gulick et al.Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), thedisclosure of which is incorporated herein by reference. Magnetic beadsare another tool that can be used to transfect hematopoietic stem cellsin a mild and efficient manner, as this methodology utilizes an appliedmagnetic field in order to direct the uptake of nucleic acids. Thistechnology is described in detail, e.g., in US 2010/0227406, thedisclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acidsby hematopoietic stem cells is laserfection, a technique that involvesexposing a cell to electromagnetic radiation of a particular wavelengthin order to gently permeabilize the cells and allow polynucleotides topenetrate the cell membrane. This technique is described in detail,e.g., in Rhodes et al. Methods in Cell Biology 82:309 (2007), thedisclosure of which is incorporated herein by reference.

Microvesicles represent another potential vehicle that can be used tomodify the genome of a hematopoietic stem cell according to the methodsof the invention described herein. For instance, microvesicles that havebeen induced by the co-overexpression of the glycoprotein VSV-G with,e.g., a genome-modifying protein, such as a nuclease, can be used toefficiently deliver proteins into a cell that subsequently catalyze thesite-specific cleavage of an endogenous polynucleotide sequence so as toprepare the genome of the cell for the covalent incorporation of apolynucleotide of interest, such as a gene or regulatory sequence. Theuse of such vesicles, also referred to as Gesicles, for the geneticmodification of eukaryotic cells is described in detail, e.g., in Quinn,T P, et al. Genetic Modification of Target Cells by Direct Delivery ofActive Protein [abstract]. In: Methylation changes in early embryonicgenes in cancer [abstract], in: Proceedings of the 18th Annual Meetingof the American Society of Gene and Cell Therapy; 2015 May 13, AbstractNo. 122.

Incorporation of Target Genes by Gene Editing Techniques

In addition to viral vectors, a variety of additional tools have beendeveloped that can be used for the incorporation of exogenous genes intohematopoietic stem cells. One such method that can be used forincorporating polynucleotides encoding target genes into hematopoieticstem cells involves the use of transposons. Transposons arepolynucleotides that encode transposase enzymes and contain apolynucleotide sequence or gene of interest flanked by 5′ and 3′excision sites. Once a transposon has been delivered into a cell,expression of the transposase gene commences and results in activeenzymes that cleave the gene of interest from the transposon. Thisactivity is mediated by the site-specific recognition of transposonexcision sites by the transposase. In certain cases, these excisionsites may be terminal repeats or inverted terminal repeats. Once excisedfrom the transposon, the gene of interest can be integrated into thegenome of a mammalian cell by transposase-catalyzed cleavage of similarexcision sites that exist within the nuclear genome of the cell. Thisallows the gene of interest to be inserted into the cleaved nuclear DNAat the complementary excision sites, and subsequent covalent ligation ofthe phosphodiester bonds that join the gene of interest to the DNA ofthe mammalian cell genome completes the incorporation process. Incertain cases, the transposon may be a retrotransposon, such that thegene encoding the target gene is first transcribed to an RNA product andthen reverse-transcribed to DNA before incorporation in the mammaliancell genome. Exemplary transposon systems include the piggybactransposon (described in detail in, e.g., WO 2010/085699) and thesleeping beauty transposon (described in detail in, e.g.,US2005/0112764), the disclosures of each of which are incorporatedherein by reference.

Another useful tool for the integration of target genes into the genomeof a hematopoietic stem cell is the clustered regularly interspacedshort palindromic repeats (CRISPR)/Cas system, a system that originallyevolved as an adaptive defense mechanism in bacteria and archaea againstviral infection. The CRISPR/Cas system includes palindromic repeatsequences within plasmid DNA and an associated Cas9 nuclease. Thisensemble of DNA and protein directs site specific DNA cleavage of atarget sequence by first incorporating foreign DNA into CRISPR loci.Polynucleotides containing these foreign sequences and the repeat-spacerelements of the CRISPR locus are in turn transcribed in a host cell tocreate a guide RNA, which can subsequently anneal to a target sequenceand localize the Cas9 nuclease to this site. In this manner, highlysite-specific cas9-mediated DNA cleavage can be engendered in a foreignpolynucleotide because the interaction that brings cas9 within closeproximity of the target DNA molecule is governed by RNA:DNAhybridization. As a result, one can theoretically design a CRISPR/Cassystem to cleave any target DNA molecule of interest. This technique hasbeen exploited in order to edit eukaryotic genomes (Hwang et al. NatureBiotechnology 31:227 (2013)) and can be used as an efficient means ofsite-specifically editing hematopoietic stem cell genomes in order tocleave DNA prior to the incorporation of a gene encoding a target gene.The use of CRISPR/Cas to modulate gene expression has been described in,e.g., U.S. Pat. No. 8,697,359, the disclosure of which is incorporatedherein by reference. Alternative methods for site-specifically cleavinggenomic DNA prior to the incorporation of a gene of interest in ahematopoietic stem cell include the use of zinc finger nucleases (ZFNs)and transcription activator-like effector nucleases (TALENs). Unlike theCRISPR/Cas system, these enzymes do not contain a guiding polynucleotideto localize to a specific target sequence. Target specificity is insteadcontrolled by DNA binding domains within these enzymes. The use of ZFNsand TALENs in genome editing applications is described, e.g., in Urnovet al. Nature Reviews Genetics 11:636 (2010); and in Joung et al. NatureReviews Molecular Cell Biology 14:49 (2013), the disclosure of both ofwhich are incorporated herein by reference.

Additional genome editing techniques that can be used to incorporatepolynucleotides encoding target genes into the genome of a hematopoieticstem cell include the use of ARCUS™ meganucleases that can be rationallydesigned so as to site-specifically cleave genomic DNA. The use of theseenzymes for the incorporation of genes encoding target genes into thegenome of a mammalian cell is advantageous in view of the definedstructure-activity relationships that have been established for suchenzymes. Single chain meganucleases can be modified at certain aminoacid positions in order to create nucleases that selectively cleave DNAat desired locations, enabling the site-specific incorporation of atarget gene into the nuclear DNA of a hematopoietic stem cell. Thesesingle-chain nucleases have been described extensively in, e.g., U.S.Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which areincorporated herein by reference.

Inducing the Differentiation of Hematopoietic Stem Cells

In certain cases, it may be desirable to expand, enrich, or maintain apopulation of hematopoietic stem cells according to the methods of theinvention and subsequently induce the differentiation of these cellsinto a blood cell of the hematopoietic repertoire prior to infusion ofthe resulting cells into a recipient. This represents a useful paradigmfor administering a specific blood cell type to a recipient in needthereof. Populations of hematopoietic stem cells that have beenexpanded, enriched, and/or maintained according to the methods of theinvention may be subjected to various conditions in order to stimulatethe differentiation of these cells into cells of the hematopoieticlineage, such as conditions that are known in the art. For instance,using established protocols, hematopoietic stem cells can be induced todifferentiate into one of a multitude of blood cell types, such ascommon lymphoid progenitor cells, common myeloid progenitor cells,megakaryocyte-erythroid progenitor cells, granulocyte-megakaryocyteprogenitor cells, granulocytes, promyelocytes, neutrophils, eosinophils,basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,platelet-producing megakaryocytes, platelets, monocytes, macrophages,dendritic cells, microglia, osteoclasts, and lymphocytes, such as NKcells, B-cells and T-cells.

Indications for Hematopoietic Stem Cell Therapy

Hematopoietic stem cells produced (e.g., expanded, enriched, ormaintained in a multi-potent state) through the use of the compositionsand methods of the invention can be used to treat a variety of humandiseases. Hematopoietic stem cells or progeny thereof administered to apatient may be autologous, syngeneic, or allogeneic, and may beadministered in conjunction with one or more agents that promote theexpansion of a hematopoietic stem cell in vivo. For instance,hematopoietic stem cells or progeny thereof may be administered to apatient (e.g., a human patient) in order to treat such diseases as AcuteLymphoblastic Leukemia (ALL), Acute Myelogenous Leukemia (AML), ChronicMyelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), HodgkinLymphoma (HL), Non-Hodgkin Lymphoma (NHL), Myelodysplastic Syndrome(MDS), Multiple myeloma, Aplastic anemia, Bone marrow failure,Myeloproliferative disorders such as Myelofibrosis, Essentialthrombocytopenia or Polycythemia vera, Fanconi anemia, Dyskeratosiscongenita, Common variable immune deficiency (CVID, such as CVID 1, CVID2, CVID 3, CVID 4, CVID 5, and CVID 6), Human immunodeficiency virus(HIV), Hemophagocytic lymphohistiocystosis, Amyloidosis, Solid tumorssuch as Neuroblastoma, Germ cell tumors, Breast cancer, Wilms' tumor,Medulloblastoma, and Neuroectodermal tumors, Autoimmune diseases such asScleroderma, Multiple sclerosis, Ulcerative colitis, Systemic lupuserythematosus and Type I diabetes, or protein deficiencies such asAdrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MLD),Hemophilia A & B, Hurler syndrome, Hunter syndrome, Fabry disease,Gaucher disease, Epidermolysis bullosa, Globoid Cell Leukodystrophy,Sanfillipo syndrome, and Morquio syndrome.

Hematopoietic stem cells or progeny thereof can also be administered toa human patient in order to treat a genetic blood disorder, such asSickle cell anemia, Alpha thalassemia, Beta thalassemia, Deltathalassemia, Hemoglobin E/thalassemia, Hemoglobin S/thalassemia,Hemoglobin C/thalassemia, Hemoglobin D/thalassemia, Chronicgranulomatous disease (X-linked Chronic granulomatous disease, autosomalrecessive (AR) chronic granulomatous disease, chronic granulomatousdisease AR I NCF1, Chronic granulomatous disease AR CYBA, Chronicgranulomatous disease AR II NCF2, Chronic granulomatous disease AR IIINCF4), X-linked Severe Combined Immune Deficiency (SCID), ADA SCID,IL7-RA SCID, CD3 SCID, Rag1/Rag2 SCID, Artemis SCID, CD45 SCID, Jak3SCID, Congenital agranulocytosis, Congenital agranulocytosis-congenitalneutropenia-SCN1, Congenital agranulocytosis-congenitalneutropenia-SCN2, Familial hemophagocytic lymphohistiocystosis (FHL),Familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforinmutation), Agammaglobulinemia (X-linked Agammaglobulinemia),Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, Hemolytic anemia dueto red cell pyruvate kinase deficiency, Paroxysmal nocturnalhemoglobinuria, X-linked Adrenoleukodystrophy (X-ALD), X-linkedlymphoproliferative disease, Unicentric Castleman's Disease,Multicentric Castleman's Disease, Congenital amegakaryocyticthrombocytopenia (CAMT) type I, Reticular dysgenesis, Fanconi anemia,Acquired idiopathic sideroblastic anemia, Systemic mastocytosis, Vonwillebrand disease (VWD), Congenital dyserythropoietic anemia type 2,Cartilage-hair hypoplasia syndrome, Hereditary spherocytosis,Blackfan-Diamond syndrome, Shwachman-Diamond syndrome,Thrombocytopenia-absent radius syndrome, Osteopetrosis, Infantileosteopetrosis, Mucopolysaccharidoses, Lesch-Nyhan syndrome, Glycogenstorage disease, Congenital mastocytosis, Omenn syndrome, X-linkedImmunodysregulation, polyendocrinopathy, and enteropathy (IPEX), IPEXcharacterized by mutations in FOXP3, X-linked syndrome ofpolyendocrinopathy, immune dysfunction, and diarrhea (XPID), X-LinkedAutoimmunity-Allergic Dysregulation Syndrome (XLAAD), IPEX-likesyndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3, HyperIgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin M, Barelymphocyte Syndrome type I, and Bare lymphocyte Syndrome type II (Barelymphocyte Syndrome type II, MHC class I deficiency; Bare lymphocyteSyndrome type II, complementation group A; Bare lymphocyte Syndrome typeII, complementation group C; Bare lymphocyte Syndrome type IIcomplementation group D; Bare lymphocyte Syndrome type II,complementation group E). Populations of hematopoietic stem cellsexpanded, enriched, or maintained by the compositions and/or methods ofthe invention, as well as progeny thereof, can also be used to treat apatient suffering from a hematolymphoid malignancy, a non-hematolymphoidmalignancy, or a protein deficiency. In other embodiments, the patientmay be tissue or cell transplantation recipient, and the hematopoieticstem cells or progeny thereof are administered in order to inducetolerance to the transplanted tissue or cells.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications, cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with one or more agents that together exhibittwo or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

2. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together exhibit two or more activitiesselected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

3. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together exhibittwo or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

4. The method of any one of paragraphs 1-3, wherein the modulation ofhistone methylation is activation of histone methylation, maintenance ofhistone methylation, or inhibition of histone demethylation.

5. The method of any one of paragraphs 1-4, wherein the modulation ofhistone acetylation is activation of histone acetylation, maintenance ofhistone acetylation, or inhibition of histone deacetylation.

6. The method of paragraph 4, wherein said one or more agents comprise acompound that activates histone methylation, maintains histonemethylation, or inhibits histone demethylation and a compound thatinhibits TGFβ signaling.

7. The method of paragraph 6, wherein said compound that activateshistone methylation, maintains histone methylation, or inhibits histonedemethylation is a histone demethylase inhibitor and said compound thatinhibits TGFβ signaling is a TGFβ receptor inhibitor.8. The method of paragraph 7, wherein said histone demethylase inhibitoris a LSD1 inhibitor.9. The method of paragraph 8, wherein said LSD1 inhibitor is LSD1inhibitor IV RN-1 and said TGFβ receptor inhibitor is ALK5 inhibitor II.10. The method of paragraph 8, wherein said LSD1 inhibitor istranylcypromine and said TGFβ receptor inhibitor is ALK5 inhibitor II.11. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

12. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together inhibit the activity of two ormore proteins selected from the group consisting of:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

13. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

14. The method of any one of paragraphs 11-13, wherein the one or moreagents comprise a combination of agents selected from the combination ofagents of Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6.

15. The method of any one of paragraphs 11-13, wherein said histonedemethylase is LSD1.

16. The method of any one of paragraphs 11-13, wherein said one or moreagents comprise a histone demethylase inhibitor selected from the groupconsisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine.17. The method of any one of paragraphs 11-16, wherein said protein thatpropagates TGFβ signaling is a TGFβ receptor.18. The method of any one of paragraphs 11-17, wherein said one or moreagents comprise a compound that inhibits a protein that propagates TGFβsignaling selected from the group consisting of ALK5 inhibitor II,LY364947, A83-01, and DMH1.19. The method of any one of paragraphs 11-18, wherein said one or moreagents comprise a compound that inhibits a protein that propagates p38signaling, and wherein said compound is SB203580.20. The method of any one of paragraphs 11-19, wherein said one or moreagents comprise a compound that inhibits a protein that promotesβ-catenin degradation selected from the group consisting of CHIR99021,lithium chloride, BIO, and FGF2.21. The method of any one of paragraphs 11-20, wherein said one or moreagents comprise a compound that inhibits a histone deacetylase areselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.22. The method of any one of paragraphs 11-21, wherein said one or moreagents together inhibit the activity of a histone demethylase and aprotein that propagates TGFβ signaling.23. The method of paragraph 22, wherein said histone demethylase isLSD1.24. The method of paragraph 22 or 23, wherein said protein thatpropagates TGFβ signaling is a TGFβ receptor.25. The method of any one of paragraphs 22-24, wherein said one or moreagents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II.26. The method of any one of paragraphs 22-25, wherein said one or moreagents comprise a compound that inhibits p38 signaling.27. The method of any one of paragraphs 22-26, wherein said one or moreagents comprise a compound that inhibits a histone deacetylase.28. The method of any one of paragraphs 22-27, wherein said one or moreagents further comprise a compound that inhibits BMP signaling.29. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with (a) a first agent selected from the groupconsisting of an LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine, and (b) a second agent selected from the groupconsisting of ALK5 inhibitor II, LY364947, A83-01, Trichostatin A,SB203580, CHIR99021, DMH1, sodium acetate, and istodax.30. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith (a) a first agent selected from the group consisting of an LSD1inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76,LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and Tranylcypromine, and(b) a second agent selected from the group consisting of ALK5 inhibitorII, LY364947, A83-01, Trichostatin A, SB203580, CHIR99021, DMH1, sodiumacetate, and istodax.31. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with said method comprising contacting apopulation of hematopoietic stem cells with (a) a first agent selectedfrom the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor IIS2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1inhibitor I, and Tranylcypromine, and (b) a second agent selected fromthe group consisting of ALK5 inhibitor II, LY364947, A83-01,Trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax,wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said firstand second agents.32. The method of any one of paragraphs 29-31, wherein the one or moreagents comprise a combination of agents selected from the combination ofagents of Table 7, Table 8, Table 9, and Table 10.33. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to stimulate expansionof said population of cells by 10% or more relative to a population ofhematopoietic stem cells not contacted with said one or more agentsafter seven or more days of culture (e.g., after seven, ten, twelve,fourteen, fifteen, twenty, or more days of culture).34. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to stimulate expansionof said population of cells by 10% or more relative to a population ofhematopoietic stem cells that have been contacted with a substance thatinhibits aryl hydrocarbon receptor signaling such as SR1 or an analogthereof, UM171 or an analog thereof, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1 such as nicotinamide,cambinol, or an analog thereof, after seven or more days of culture(e.g., after seven, ten, twelve, fourteen, fifteen, twenty, or more daysof culture).35. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells not contacted withsaid one or more agents after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).36. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells that have beencontacted with a substance that inhibits aryl hydrocarbon receptorsignaling such as SR1 or an analog thereof, UM171 or an analog thereof,a prostaglandin, an agonist of Notch signaling, or an inhibitor of SIRT1such as nicotinamide, cambinol, or an analog thereof, after seven ormore days of culture (e.g., after seven, ten, twelve, fourteen, fifteen,twenty, or more days of culture).37. The method of any one of paragraphs 3, 13, and 31, wherein saidfirst population of hematopoietic stem cells exhibits a hematopoieticstem cell functional potential after three or more days of culture(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty, ormore days) that is greater than that of said control population ofhematopoietic stem cells.38. The method of any one of paragraphs 1-37, wherein said hematopoieticstem cells are mammalian cells.39. The method of paragraph 38, wherein said mammalian cells are humancells.40. The method of paragraph 39, wherein said hematopoietic stem cellsare CD34+ cells.41. The method of paragraph 40, wherein at least 10% of said CD34+ cellsare CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+ cells.42. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human cord blood.43. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human mobilized peripheral blood.44. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human bone marrow.45. The method of any one of paragraphs 38-44, wherein saidhematopoietic stem cells are freshly isolated from said human.46. The method of any one of paragraphs 38-44, wherein saidhematopoietic stem cells have been previously cryopreserved.47. The method of paragraph 38, wherein said mammalian cells are murinecells.48. The method of any one of paragraphs 1-47, wherein said hematopoieticstem cells are cultured for two or more days (e.g., three, five, seven,ten, twelve, fourteen, fifteen, twenty, or more days).49. The method of any one of paragraphs 1-48, wherein said hematopoieticstem cells contact said one or more agents for two or more days (e.g.,three, five, seven, ten, twelve, fourteen, fifteen, twenty, or moredays).50. The method of any one of paragraphs 1-49, wherein said hematopoieticstem cells are contacted with said one or more agents simultaneously.51. The method of any one of paragraphs 1-49, wherein said hematopoieticstem cells are contacted with said one or more agents at differenttimes.52. The method of any one of paragraphs 1-51, wherein said hematopoieticstem cells maintain hematopoietic stem cell functional potential aftertwo days (e.g., three, five, seven, ten, twelve, fourteen, fifteen,twenty, or more days) in culture.53. The method of paragraph 52, wherein said hematopoietic stem cellsmaintain hematopoietic stem cell functional potential followingtransplantation after two days (e.g., three, five, seven, ten, twelve,fourteen, fifteen, twenty, or more days) in culture.54. The method of any one of paragraphs 1-53, wherein said hematopoieticstem cells maintain long term engraftment potential after two days(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty, ormore days) in culture.55. The method of any one of paragraphs 1-54, wherein upontransplantation into a patient, said hematopoietic stem cells give riseto recovery of a population of cells selected from the group consistingof neutrophils, platelets, red blood cells, monocytes, macrophages,antigen-presenting cells, microglia, osteoclasts, dendritic cells, andlymphocytes.56. The method of paragraph 55, wherein said lymphocytes are selectedfrom the group consisting of natural killer cells, T cells (e.g., CD4+or CD8+ cells), and B cells.57. The method of any one of paragraphs 1-56, wherein said hematopoieticstem cells are capable of localizing to hematopoietic tissue toreestablish productive hematopoiesis in a transplanted recipient.58. The method of any one of paragraphs 1-57, wherein said hematopoieticstem cells are cultured on a plastic surface or on a substrate thatincludes vitronectin, fibronectin, or matrigel.59. The method of any one of paragraphs 1-58, wherein said hematopoieticstem cells are cultured in the presence of 2-20% oxygen.60. The method of paragraph 59, wherein said hematopoietic stem cellsare cultured in the presence of 2-12% oxygen.61. The method of paragraph 60, wherein said hematopoietic stem cellsare cultured in the presence of about 5% oxygen.62. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within a mononuclear cell fraction prior totreatment with said one or more agents.63. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within a CD34+, CD34+CD38-, CD34+CD38-CD90+,CD34+CD38-CD90+CD45RA−, or CD34+CD38-CD90+CD45RA-CD49F+ enriched cellfraction prior to contacting said one or more agents.64. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within an un-enriched cell fraction prior tocontacting said one or more agents.65. A method of introducing a polynucleotide into a population ofhematopoietic stem cells, said method comprising:

-   -   a. inserting the polynucleotide into said population of        hematopoietic stem cells; and    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 1, 11, 29, or        maintaining the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells according to the        method of any one of paragraphs 3, 13, and 31-64.        66. The method of paragraph 65, wherein (a) precedes (b).        67. The method of paragraph 65, wherein (b) precedes (a).        68. The method of any one of paragraphs 65-67, wherein said        method comprises providing one or more reagents that cleave a        nucleic acid in said cells.        69. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a zinc finger        nuclease.        70. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a        transcription activator-like effector nuclease.        71. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a        CRISPR-associated protein.        72. The method of paragraph 68, wherein the one or more agents        that cleave a nucleic acid in said cells comprise a        meganuclease.        73. The method of any one of paragraphs 65-72, wherein said        method comprises contacting the hematopoietic stem cells with a        vector selected from the group consisting of a viral vector        (such as retrovirus, adenovirus, parvovirus, coronavirus,        rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpes        virus, or poxvirus) and a transposable element (such as a        piggybac transposon or sleeping beauty transposon)        74. The method of any one of paragraphs 65-72, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by electroporation, Nucleofection™, or        squeeze-poration.        75. The method of any one of paragraphs 65-72, wherein said        method comprises contacting the cells with a transformation        agent selected from the group consisting of a cationic polymer        (e.g., diethylaminoethyl-dextran), a cationic lipid, calcium        phosphate, an activated dendrimer, and a magnetic bead.        76. The method of any one of paragraphs 65-72, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by microinjection or laserfection.        77. The method of any one of paragraphs 65-76, wherein said        polynucleotide comprises a regulatory sequence selected from the        group consisting of a promoter, enhancer, or silencer sequence.        78. The method of any one of paragraphs 65-76, wherein said        polynucleotide encodes a molecule selected from the group        consisting of a protein and a RNA (mRNA, tRNA, siRNA, miRNA,        shRNA).        79. The method of any one of paragraphs 65-76, wherein said        polynucleotide is a chemically modified RNA.        80. The method of any one of paragraphs 65-79, wherein said        method further comprises introducing the population of expanded        hematopoietic stem cells or progeny thereof into a recipient.        81. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 1, 11, 29, and        32-64;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of expanded hematopoietic stem        cells or progeny thereof into said recipient.        82. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. enriching said population of hematopoietic stem cells        according to the method of any one of paragraphs 2, 12, 30, and        32-64;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of cells enriched with        hematopoietic stem cells or progeny thereof into said recipient.        83. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. maintaining the hematopoietic stem cell functional potential        of said population of hematopoietic stem cells according to the        method of any one of paragraphs 3, 13, and 31-64; c. optionally        differentiating said hematopoietic stem cells into common        lymphoid progenitor cells, common myeloid progenitor cells,        megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        84. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells produced        by the method of any one of paragraphs 1-64;    -   b. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   c. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        85. The method of any one of paragraphs 80-84, wherein said        recipient is a human.        86. The method of paragraph 85, wherein said hematopoietic stem        cells are derived from one or more hematopoietic stem cells        isolated from a human donor.        87. The method of paragraph 86, wherein said hematopoietic stem        cells are from mobilized peripheral blood of said donor.        88. The method of paragraph 87, wherein said donor has been        previously administered one or more mobilizing agents selected        from the group consisting of a CXCR4 antagonist (e.g., AMD3100),        GCSF, and GROβ.        89. The method of any one of paragraphs 1-88, wherein the        hematopoietic stem cells are additionally contacted with a        substance that inhibits aryl hydrocarbon receptor signaling.        90. The method of any one of paragraphs 34, 36, and 89, wherein        the substance that inhibits aryl hydrocarbon receptor signaling        is SR1 or an analog thereof.        91. The methods of any one of paragraphs 1-90, wherein the        hematopoietic stem cells are additionally contacted with UM171        or an analog thereof.        92. The method of any one of paragraphs 1-91, wherein the        hematopoietic stem cells are additionally contacted with a        prostaglandin        93. The method of paragraph 92, wherein said prostaglandin is        dmPGE2 or an analog thereof.        94. The method of any one of paragraphs 1-93, wherein the        hematopoietic stem cells are additionally contacted with an        agonist of Notch signaling.        95. The method of any one of paragraphs 1-94, wherein the        hematopoietic stem cells are additionally contacted with an        inhibitor of SIRT1.        96. The method of paragraph 95, wherein said inhibitor or SIRT1        is selected from the group consisting of nicotinamide, cambinol,        and analogs thereof.        97. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Acute Lymphoblastic Leukemia (ALL),        Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia        (CML), Chronic Lymphocytic Leukemia (CLL), Hodgkin Lymphoma        (HL), Non-Hodgkin Lymphoma (NHL), Myelodysplastic Syndrome        (MDS), Multiple myeloma, Aplastic anemia, Bone marrow failure,        Myeloproliferative disorders such as Myelofibrosis, Essential        thrombocytopenia or Polycythemia vera, Fanconi anemia,        Dyskeratosis congenita, Common variable immune deficiency (CVID,        such as CVID 1, CVID 2, CVID 3, CVID 4, CVID 5, and CVID 6),        Human immunodeficiency virus (HIV), Hemophagocytic        lymphohistiocystosis, Amyloidosis, Solid tumors such as        Neuroblastoma, Germ cell tumors, Breast cancer, Wilms' tumor,        Medulloblastoma, and Neuroectodermal tumors, Autoimmune diseases        such as Scleroderma, Multiple sclerosis, Ulcerative colitis,        Systemic lupus erythematosus and Type I diabetes, or protein        deficiencies such as Adrenoleukodystrophy (ALD), Metachromatic        leukodystrophy (MLD), Hemophilia A & B, Hurler syndrome, Hunter        syndrome, Fabry disease, Gaucher disease, Epidermolysis bullosa,        Globoid Cell Leukodystrophy, Sanfillipo syndrome, and Morquio        syndrome.        98. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Sickle cell anemia, Alpha        thalassemia, Beta thalassemia, Delta thalassemia, Hemoglobin        E/thalassemia, Hemoglobin S/thalassemia, Hemoglobin        C/thalassemia, Hemoglobin D/thalassemia, Chronic granulomatous        disease (X-linked Chronic granulomatous disease, autosomal        recessive (AR) chronic granulomatous disease, chronic        granulomatous disease AR I NCF1, Chronic granulomatous disease        AR CYBA, Chronic granulomatous disease AR II NCF2, Chronic        granulomatous disease AR III NCF4), X-linked Severe Combined        Immune Deficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID,        Rag1/Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, Congenital        agranulocytosis, Congenital agranulocytosis-congenital        neutropenia-SCN1, Congenital agranulocytosis-congenital        neutropenia-SCN2, Familial hemophagocytic lymphohistiocystosis        (FHL), Familial hemophagocytic lymphohistiocytosis type 2 (FHL2,        perforin mutation), Agammaglobulinemia (X-linked        Agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi        syndrome, Hemolytic anemia due to red cell pyruvate kinase        deficiency, Paroxysmal nocturnal hemoglobinuria, X-linked        Adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative        disease, Unicentric Castleman's Disease, Multicentric        Castleman's Disease, Congenital amegakaryocytic thrombocytopenia        (CAMT) type I, Reticular dysgenesis, Fanconi anemia, Acquired        idiopathic sideroblastic anemia, Systemic mastocytosis, Von        willebrand disease (VWD), Congenital dyserythropoietic anemia        type 2, Cartilage-hair hypoplasia syndrome, Hereditary        spherocytosis, Blackfan-Diamond syndrome, Shwachman-Diamond        syndrome, Thrombocytopenia-absent radius syndrome,        Osteopetrosis, Infantile osteopetrosis, Mucopolysaccharidoses,        Lesch-Nyhan syndrome, Glycogen storage disease, Congenital        mastocytosis, Omenn syndrome, X-linked Immunodysregulation,        polyendocrinopathy, and enteropathy (IPEX), IPEX characterized        by mutations in FOXP3, X-linked syndrome of polyendocrinopathy,        immune dysfunction, and diarrhea (XPID), X-Linked        Autoimmunity-Allergic Dysregulation Syndrome (XLAAD), IPEX-like        syndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3,        Hyper IgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin        M, Bare lymphocyte Syndrome type I, and Bare lymphocyte Syndrome        type II (Bare lymphocyte Syndrome type II, MHC class I        deficiency; Bare lymphocyte Syndrome type II, complementation        group A; Bare lymphocyte Syndrome type II, complementation group        C; Bare lymphocyte Syndrome type II complementation group D;        Bare lymphocyte Syndrome type II, complementation group E).        99. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a hematolymphoid        malignancy, a non-hematolymphoid malignancy, or a protein        deficiency, or a tissue or cell transplantation recipient (e.g.,        to induce tolerance to transplanted tissue or cells).        100. The method of any one of paragraphs 80-99, wherein said        hematopoietic stem cells are autologous or syngeneic.        101. The method of any one of paragraphs 80-99, wherein said        hematopoietic stem cells are allogeneic.        102. A composition comprising one or more agents that together        exhibit two or more activities selected from the group        consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation.        103. The composition of paragraph 102, wherein the modulation of        histone methylation is activation of histone methylation,        maintenance of histone methylation, or inhibition of histone        demethylation.        104. The composition of paragraph 102 or 103, wherein the        modulation of histone acetylation is activation of histone        acetylation, maintenance of histone acetylation, or inhibition        of histone deacetylation.        105. The composition of paragraph 103, wherein said one or more        agents comprise a compound that activates histone methylation,        maintains histone methylation, or inhibits histone demethylation        and a compound that inhibits TGFβ signaling.        106. The composition of paragraph 105, wherein said compound        that activates histone methylation, maintains histone        methylation, or inhibits histone demethylation a histone        demethylase inhibitor and said compound that inhibits TGFβ        signaling is a TGFβ receptor inhibitor.        107. The composition of paragraph 106, wherein said histone        demethylase inhibitor is a LSD1 inhibitor.        108. The composition of paragraph 107, wherein said LSD1        inhibitor is LSD1 inhibitor IV RN-1 and said TGFβ receptor        inhibitor is ALK5 inhibitor II.        109. The composition of paragraph 107, wherein said LSD1        inhibitor is tranylcypromine and said TGFβ receptor inhibitor is        ALK5 inhibitor II.        110. A composition comprising one or more agents that together        inhibit the activity of two or more proteins selected from the        group consisting of:    -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase.        111. The composition of paragraph 110, wherein said histone        demethylase is LSD1.        112. The composition of paragraph 110, wherein said one or more        agents comprise a histone demethylase inhibitor selected from        the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor        II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007,        LSD1 inhibitor I, and Tranylcypromine.        113. The composition of any one of paragraphs 110-112, wherein        said protein that propagates TGFβ signaling is a TGFβ receptor.        114. The composition of any one of paragraphs 110-113, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates TGFβ signaling selected from the group        consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1.        115. The composition of any one of paragraphs 110-114, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates p38 signaling, and wherein said compound        is SB203580.        116. The composition of any one of paragraphs 110-115, wherein        said one or more agents comprise a compound that inhibits a        protein that promotes β-catenin degradation selected from the        group consisting of CHIR99021, lithium chloride, BIO, and FGF2.        117. The composition of any one of paragraphs 110-116, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase selected from the group consisting of        Trichostatin A, valproic acid, butyrylhydroxamic acid, and        istodax.        118. The composition of any one of paragraphs 110-117, wherein        said one or more agents together inhibit the activity of a        histone demethylase and a protein that propagates TGFβ signaling        119. The composition of paragraph 118, wherein said histone        demethylase is LSD1.        120. The composition of paragraph 118 or 119, wherein said        protein that propagates TGFβ signaling is a TGFβ receptor.        121. The composition of any one of paragraphs 118-120, wherein        said one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5        inhibitor II.        122. The composition of any one of paragraphs 118-121, wherein        said one or more agents comprise a compound that inhibits p38        signaling.        123. The composition of any one of paragraphs 118-122, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase.        124. The composition of any one of paragraphs 118-123, wherein        said one or more agents comprise a compound that inhibits BMP        signaling.        125. A composition comprising (a) a first agent selected from        the group consisting of an LSD1 inhibitor IV RN-1, LSD1        inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III        CBB1007, LSD1 inhibitor I, and Tranylcypromine, and (b) a second        agent selected from the group consisting of ALK5 inhibitor II,        LY364947, A83-01, Trichostatin A, SB203580, CHIR99021, DMH1,        sodium acetate, and istodax.        126. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts that are        sufficient to produce an expanded population of hematopoietic        stem cells.        127. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts that are        sufficient to produce a population of cells enriched with        hematopoietic stem cells.        128. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts sufficient to        maintain the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells for at least two        days.        129. The composition of any one of paragraphs 126-128, wherein        said one or more agents are present in an aqueous solution.        130. The composition of any one of paragraphs 126-128, wherein        said one or more agents are present as a lyophilized solid.        131. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to stimulate expansion of said population of cells by        10% or more relative to a population of hematopoietic stem cells        that have been contacted with a substance that inhibits aryl        hydrocarbon receptor signaling such as SR1 or an analog thereof,        UM171 or an analog thereof, a prostaglandin, an agonist of Notch        signaling, or an inhibitor of SIRT1 such as nicotinamide,        cambinol, or an analog thereof, after seven or more days of        culture (e.g., after seven, ten, twelve, fourteen, fifteen,        twenty, or more days of culture).        132. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to enrich the population of cells with hematopoietic        stem cells by 10% or more relative to a population of        hematopoietic stem cells that have been contacted with a        substance that inhibits aryl hydrocarbon receptor signaling such        as SR1 or an analog thereof, UM171 or an analog thereof, a        prostaglandin, an agonist of Notch signaling, or an inhibitor of        SIRT1 such as nicotinamide, cambinol, or an analog thereof,        after seven or more days of culture (e.g., after seven, ten,        twelve, fourteen, fifteen, twenty, or more days of culture).        133. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to maintain long term engraftment potential of said        hematopoietic stem cells post-transplantation after having        contacted said cells in culture for two or more days (e.g.,        three, five, seven, ten, twelve, fourteen, fifteen, twenty, or        more days).        134. A composition comprising a combination of agents selected        from the combination of agents of Table 1, Table 2, Table 3,        Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, and Table        10.        135. A cell culture medium comprising the composition of any one        of paragraphs 102-134.        136. The cell culture medium of paragraph 135, wherein said cell        culture medium is substantially free of serum.        137. The composition of any one of paragraphs 102-134, wherein        said composition further comprises a population of hematopoietic        stem cells in contact with said one or more agents.        138. The composition of paragraph 137, wherein said        hematopoietic stem cells have been cultured in the presence of        said one or more agents for two or more days (e.g., three, five,        seven, ten, twelve, fourteen, fifteen, twenty, or more days).        139. A method of producing an expanded population of        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic stem cells with

(1) a first agent that exhibits one or more activities selected from thegroup consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation; and

(2) a second agent selected from the group consisting of SR1 or ananalog thereof, UM171 or an analog thereof, a prostaglandin, an agonistof Notch signaling, or an inhibitor of SIRT1, wherein the first andsecond agents are present in amounts that together are sufficient toproduce an expanded population of hematopoietic stem cells.

140. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith (1) a first agent that exhibits one or more activities selectedfrom the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation; and        (2) a second agent selected from the group consisting of SR1 or        an analog thereof, UM171 or an analog thereof, a prostaglandin,        an agonist of Notch signaling, or an inhibitor of SIRT1, wherein        the first and second agents are present in amounts that together        are sufficient to produce a population of cells enriched with        hematopoietic stem cells.        141. A method of maintaining the hematopoietic stem cell        functional potential of a population of hematopoietic stem cells        ex vivo for at least two days, said method comprising contacting        a first population of hematopoietic stem cells with

(1) a first agent that exhibits one or more activities selected from thegroup consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation; and

(2) a second agent selected from the group consisting of SR1 or ananalog thereof, UM171 or an analog thereof, a prostaglandin, an agonistof Notch signaling, or an inhibitor of SIRT1, wherein the population ofhematopoietic stem cells exhibits a hematopoietic stem cell functionalpotential after two or more days that is greater than that of a controlpopulation of hematopoietic stem cells cultured under the sameconditions and for the same time as said population of hematopoieticstem cells but not contacted with said one or more agents and said oneor more substances.

142. A population of hematopoietic stem cells produced by the method ofany one of paragraphs 1-101 and 139-141.

143. A kit comprising the composition of any one of paragraphs 102-134,137, and 138, wherein said kit further comprises a package insert.

144. The kit of paragraph 143, wherein said package insert instructs auser of said kit to expand, enrich, or maintain the hematopoietic stemcell functional potential of a population of hematopoietic stem cells exvivo.

145. The kit of paragraph 143, wherein said package insert instructssaid user to express a polynucleotide in said hematopoietic stem cells.

146. The kit of paragraph 143, wherein said package insert instructssaid user to administer said hematopoietic stem cells to a patient.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with one or more agents that together exhibittwo or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

2. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together exhibit two or more activitiesselected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

3. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together exhibittwo or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

4. The method of any one of paragraphs 1-3, wherein the modulation ofhistone methylation is activation of histone methylation, maintenance ofhistone methylation, or inhibition of histone demethylation.

5. The method of any one of paragraphs 1-4, wherein the modulation ofhistone acetylation is activation of histone acetylation, maintenance ofhistone acetylation, or inhibition of histone deacetylation.

6. The method of paragraph 4, wherein said one or more agents comprise acompound that activates histone methylation, maintains histonemethylation, or inhibits histone demethylation and a compound thatinhibits TGFβ signaling.

7. The method of paragraph 6, wherein said compound that activateshistone methylation, maintains histone methylation, or inhibits histonedemethylation is a histone demethylase inhibitor and said compound thatinhibits TGFβ signaling is a TGFβ receptor inhibitor.8. The method of paragraph 7, wherein said histone demethylase inhibitoris a LSD1 inhibitor.9. The method of paragraph 8, wherein said LSD1 inhibitor is LSD1inhibitor IV RN-1 and said TGFβ receptor inhibitor is ALK5 inhibitor II.10. The method of paragraph 8, wherein said LSD1 inhibitor istranylcypromine and said TGFβ receptor inhibitor is ALK5 inhibitor II.11. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

12. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together inhibit the activity of two ormore proteins selected from the group consisting of:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

13. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase,

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

14. The method of any one of paragraphs 11-13, wherein the one or moreagents comprise a combination of agents selected from the combination ofagents of Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6.

15. The method of any one of paragraphs 11-13, wherein said histonedemethylase is LSD1.

16. The method of any one of paragraphs 11-13, wherein said one or moreagents comprise a histone demethylase inhibitor selected from the groupconsisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine.17. The method of any one of paragraphs 11-16, wherein said protein thatpropagates TGFβ signaling is a TGFβ receptor.18. The method of any one of paragraphs 11-17, wherein said one or moreagents comprise a compound that inhibits a protein that propagates TGFβsignaling selected from the group consisting of ALK5 inhibitor II,LY364947, A83-01, and DMH1.19. The method of any one of paragraphs 11-18, wherein said one or moreagents comprise a compound that inhibits a protein that propagates p38signaling, and wherein said compound is SB203580.20. The method of any one of paragraphs 11-19, wherein said one or moreagents comprise a compound that inhibits a protein that promotesβ-catenin degradation selected from the group consisting of CHIR99021,lithium chloride, BIO, and FGF2.21. The method of any one of paragraphs 11-20, wherein said one or moreagents comprise a compound that inhibits a histone deacetylase areselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.22. The method of any one of paragraphs 11-21, wherein said one or moreagents together inhibit the activity of a histone demethylase and aprotein that propagates TGFβ signaling.23. The method of paragraph 22, wherein said histone demethylase isLSD1.24. The method of paragraph 22 or 23, wherein said protein thatpropagates TGFβ signaling is a TGFβ receptor.25. The method of any one of paragraphs 22-24, wherein said one or moreagents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II.26. The method of any one of paragraphs 22-25, wherein said one or moreagents comprise a compound that inhibits p38 signaling.27. The method of any one of paragraphs 22-26, wherein said one or moreagents comprise a compound that inhibits a histone deacetylase.28. The method of any one of paragraphs 22-27, wherein said one or moreagents further comprise a compound that inhibits BMP signaling.29. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with (a) a first agent selected from the groupconsisting of an LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine, and (b) a second agent selected from the groupconsisting of ALK5 inhibitor II, LY364947, A83-01, Trichostatin A,SB203580, CHIR99021, DMH1, sodium acetate, and istodax.30. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith (a) a first agent selected from the group consisting of an LSD1inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76,LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and Tranylcypromine, and(b) a second agent selected from the group consisting of ALK5 inhibitorII, LY364947, A83-01, Trichostatin A, SB203580, CHIR99021, DMH1, sodiumacetate, and istodax.31. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with said method comprising contacting apopulation of hematopoietic stem cells with (a) a first agent selectedfrom the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor IIS2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1inhibitor I, and Tranylcypromine, and (b) a second agent selected fromthe group consisting of ALK5 inhibitor II, LY364947, A83-01,Trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax,wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said firstand second agents.32. The method of any one of paragraphs 29-31, wherein the one or moreagents comprise a combination of agents selected from the combination ofagents of Table 7, Table 8, Table 9, and Table 10.33. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to stimulate expansionof said population of cells by 10% or more relative to a population ofhematopoietic stem cells not contacted with said one or more agentsafter seven or more days of culture (e.g., after seven, ten, twelve,fourteen, fifteen, twenty, or more days of culture).34. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to stimulate expansionof said population of cells by 10% or more relative to a population ofhematopoietic stem cells that have been contacted with a substance thatinhibits aryl hydrocarbon receptor signaling such as SR1 or an analogthereof, UM171 or an analog thereof, a UM171 analog selected from Table11, a prostaglandin, an agonist of Notch signaling, or an inhibitor ofSIRT1 such as nicotinamide, cambinol, or an analog thereof, after sevenor more days of culture (e.g., after seven, ten, twelve, fourteen,fifteen, twenty, or more days of culture).35. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells not contacted withsaid one or more agents after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).36. The method of any one of paragraphs 1-32, wherein said one or moreagents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells that have beencontacted with a substance that inhibits aryl hydrocarbon receptorsignaling such as SR1 or an analog thereof, UM171 or an analog thereof,a UM171 analog selected from Table 11, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1 such as nicotinamide,cambinol, or an analog thereof, after seven or more days of culture(e.g., after seven, ten, twelve, fourteen, fifteen, twenty, or more daysof culture).37. The method of any one of paragraphs 3, 13, and 31, wherein saidfirst population of hematopoietic stem cells exhibits a hematopoieticstem cell functional potential after three or more days of culture(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty, ormore days) that is greater than that of said control population ofhematopoietic stem cells.38. The method of any one of paragraphs 1-37, wherein said hematopoieticstem cells are mammalian cells.39. The method of paragraph 38, wherein said mammalian cells are humancells.40. The method of paragraph 39, wherein said hematopoietic stem cellsare CD34+ cells.41. The method of paragraph 40, wherein at least 10% of said CD34+ cellsare CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+ cells.42. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human cord blood.43. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human mobilized peripheral blood.44. The method of any one of paragraphs 38-40, wherein saidhematopoietic stem cells are from human bone marrow.45. The method of any one of paragraphs 38-44, wherein saidhematopoietic stem cells are freshly isolated from said human.46. The method of any one of paragraphs 38-44, wherein saidhematopoietic stem cells have been previously cryopreserved.47. The method of paragraph 38, wherein said mammalian cells are murinecells.48. The method of any one of paragraphs 1-47, wherein said hematopoieticstem cells are cultured for two or more days (e.g., three, five, seven,ten, twelve, fourteen, fifteen, twenty, or more days).49. The method of any one of paragraphs 1-48, wherein said hematopoieticstem cells contact said one or more agents for two or more days (e.g.,three, five, seven, ten, twelve, fourteen, fifteen, twenty, or moredays).50. The method of any one of paragraphs 1-49, wherein said hematopoieticstem cells are contacted with said one or more agents simultaneously.51. The method of any one of paragraphs 1-49, wherein said hematopoieticstem cells are contacted with said one or more agents at differenttimes.52. The method of any one of paragraphs 1-51, wherein said hematopoieticstem cells maintain hematopoietic stem cell functional potential aftertwo days (e.g., three, five, seven, ten, twelve, fourteen, fifteen,twenty, or more days) in culture.53. The method of paragraph 52, wherein said hematopoietic stem cellsmaintain hematopoietic stem cell functional potential followingtransplantation after two days (e.g., three, five, seven, ten, twelve,fourteen, fifteen, twenty, or more days) in culture.54. The method of any one of paragraphs 1-53, wherein said hematopoieticstem cells maintain long term engraftment potential after two days(e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty, ormore days) in culture.55. The method of any one of paragraphs 1-54, wherein upontransplantation into a patient, said hematopoietic stem cells give riseto recovery of a population of cells selected from the group consistingof neutrophils, platelets, red blood cells, monocytes, macrophages,antigen-presenting cells, microglia, osteoclasts, dendritic cells, andlymphocytes.56. The method of paragraph 55, wherein said lymphocytes are selectedfrom the group consisting of natural killer cells, T cells (e.g., CD4+or CD8+ cells), and B cells.57. The method of any one of paragraphs 1-56, wherein said hematopoieticstem cells are capable of localizing to hematopoietic tissue toreestablish productive hematopoiesis in a transplanted recipient.58. The method of any one of paragraphs 1-57, wherein said hematopoieticstem cells are cultured on a plastic surface or on a substrate thatincludes vitronectin, fibronectin, or matrigel.59. The method of any one of paragraphs 1-58, wherein said hematopoieticstem cells are cultured in the presence of 2-20% oxygen.60. The method of paragraph 59, wherein said hematopoietic stem cellsare cultured in the presence of 2-12% oxygen.61. The method of paragraph 60, wherein said hematopoietic stem cellsare cultured in the presence of about 5% oxygen.62. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within a mononuclear cell fraction prior totreatment with said one or more agents.63. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within a CD34+, CD34+CD38-, CD34+CD38-CD90+,CD34+CD38-CD90+CD45RA−, or CD34+CD38-CD90+CD45RA-CD49F+ enriched cellfraction prior to contacting said one or more agents.64. The method of any one of paragraphs 1-61, wherein said hematopoieticstem cells are originally within an un-enriched cell fraction prior tocontacting said one or more agents.65. A method of introducing a polynucleotide into a population ofhematopoietic stem cells, said method comprising:

-   -   a. inserting the polynucleotide into said population of        hematopoietic stem cells; and    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 1, 11, 29, or        maintaining the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells according to the        method of any one of paragraphs 3, 13, and 31-64.        66. The method of paragraph 65, wherein (a) precedes (b).        67. The method of paragraph 65, wherein (b) precedes (a).        68. The method of any one of paragraphs 65-67, wherein said        method comprises providing one or more reagents that cleave a        nucleic acid in said cells.        69. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a zinc finger        nuclease.        70. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a        transcription activator-like effector nuclease.        71. The method of paragraph 68, wherein the one or more reagents        that cleave a nucleic acid in said cells comprise a        CRISPR-associated protein.        72. The method of paragraph 68, wherein the one or more agents        that cleave a nucleic acid in said cells comprise a        meganuclease.        73. The method of any one of paragraphs 65-72, wherein said        method comprises contacting the hematopoietic stem cells with a        vector selected from the group consisting of a viral vector        (such as retrovirus, adenovirus, parvovirus, coronavirus,        rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpes        virus, or poxvirus) and a transposable element (such as a        piggybac transposon or sleeping beauty transposon)        74. The method of any one of paragraphs 65-72, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by electroporation, Nucleofection™, or        squeeze-poration.        75. The method of any one of paragraphs 65-72, wherein said        method comprises contacting the cells with a transformation        agent selected from the group consisting of a cationic polymer        (e.g., diethylaminoethyl-dextran), a cationic lipid, calcium        phosphate, an activated dendrimer, and a magnetic bead.        76. The method of any one of paragraphs 65-72, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by microinjection or laserfection.        77. The method of any one of paragraphs 65-76, wherein said        polynucleotide comprises a regulatory sequence selected from the        group consisting of a promoter, enhancer, or silencer sequence.        78. The method of any one of paragraphs 65-76, wherein said        polynucleotide encodes a molecule selected from the group        consisting of a protein and a RNA (mRNA, tRNA, siRNA, miRNA,        shRNA).        79. The method of any one of paragraphs 65-76, wherein said        polynucleotide is a chemically modified RNA.        80. The method of any one of paragraphs 65-79, wherein said        method further comprises introducing the population of expanded        hematopoietic stem cells or progeny thereof into a recipient.        81. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 1, 11, 29, and        32-64;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and d.        introducing the population of expanded hematopoietic stem cells        or progeny thereof into said recipient.        82. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. enriching said population of hematopoietic stem cells        according to the method of any one of paragraphs 2, 12, 30, and        32-64;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of cells enriched with        hematopoietic stem cells or progeny thereof into said recipient.        83. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. maintaining the hematopoietic stem cell functional potential        of said population of hematopoietic stem cells according to the        method of any one of paragraphs 3, 13, and 31-64; c. optionally        differentiating said hematopoietic stem cells into common        lymphoid progenitor cells, common myeloid progenitor cells,        megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        84. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells produced        by the method of any one of paragraphs 1-64;    -   b. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   c. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        85. The method of any one of paragraphs 80-84, wherein said        recipient is a human.        86. The method of paragraph 85, wherein said hematopoietic stem        cells are derived from one or more hematopoietic stem cells        isolated from a human donor.        87. The method of paragraph 86, wherein said hematopoietic stem        cells are from mobilized peripheral blood of said donor.        88. The method of paragraph 87, wherein said donor has been        previously administered one or more mobilizing agents selected        from the group consisting of a CXCR4 antagonist (e.g., AMD3100),        GCSF, and GROβ.        89. The method of any one of paragraphs 1-88, wherein the        hematopoietic stem cells are additionally contacted with a        substance that inhibits aryl hydrocarbon receptor signaling.        90. The method of any one of paragraphs 34, 36, and 89, wherein        the substance that inhibits aryl hydrocarbon receptor signaling        is SR1 or an analog thereof.        91. The methods of any one of paragraphs 1-90, wherein the        hematopoietic stem cells are additionally contacted with UM171,        an analog thereof, or a UM171 analog selected from Table 11.        92. The method of any one of paragraphs 1-91, wherein the        hematopoietic stem cells are additionally contacted with a        prostaglandin        93. The method of paragraph 92, wherein said prostaglandin is        dmPGE2 or an analog thereof.        94. The method of any one of paragraphs 1-93, wherein the        hematopoietic stem cells are additionally contacted with an        agonist of Notch signaling.        95. The method of any one of paragraphs 1-94, wherein the        hematopoietic stem cells are additionally contacted with an        inhibitor of SIRT1.        96. The method of paragraph 95, wherein said inhibitor or SIRT1        is selected from the group consisting of nicotinamide, cambinol,        and analogs thereof.        97. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Acute Lymphoblastic Leukemia (ALL),        Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia        (CML), Chronic Lymphocytic Leukemia (CLL), Hodgkin Lymphoma        (HL), Non-Hodgkin Lymphoma (NHL), Myelodysplastic Syndrome        (MDS), Multiple myeloma, Aplastic anemia, Bone marrow failure,        Myeloproliferative disorders such as Myelofibrosis, Essential        thrombocytopenia or Polycythemia vera, Fanconi anemia,        Dyskeratosis congenita, Common variable immune deficiency (CVID,        such as CVID 1, CVID 2, CVID 3, CVID 4, CVID 5, and CVID 6),        Human immunodeficiency virus (HIV), Hemophagocytic        lymphohistiocystosis, Amyloidosis, Solid tumors such as        Neuroblastoma, Germ cell tumors, Breast cancer, Wilms' tumor,        Medulloblastoma, and Neuroectodermal tumors, Autoimmune diseases        such as Scleroderma, Multiple sclerosis, Ulcerative colitis,        Systemic lupus erythematosus and Type I diabetes, or protein        deficiencies such as Adrenoleukodystrophy (ALD), Metachromatic        leukodystrophy (MLD), Hemophilia A & B, Hurler syndrome, Hunter        syndrome, Fabry disease, Gaucher disease, Epidermolysis bullosa,        Globoid Cell Leukodystrophy, Sanfillipo syndrome, and Morquio        syndrome.        98. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Sickle cell anemia, Alpha        thalassemia, Beta thalassemia, Delta thalassemia, Hemoglobin        E/thalassemia, Hemoglobin S/thalassemia, Hemoglobin        C/thalassemia, Hemoglobin D/thalassemia, Chronic granulomatous        disease (X-linked Chronic granulomatous disease, autosomal        recessive (AR) chronic granulomatous disease, chronic        granulomatous disease AR I NCF1, Chronic granulomatous disease        AR CYBA, Chronic granulomatous disease AR II NCF2, Chronic        granulomatous disease AR III NCF4), X-linked Severe Combined        Immune Deficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID,        Rag1/Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, Congenital        agranulocytosis, Congenital agranulocytosis-congenital        neutropenia-SCN1, Congenital agranulocytosis-congenital        neutropenia-SCN2, Familial hemophagocytic lymphohistiocystosis        (FHL), Familial hemophagocytic lymphohistiocytosis type 2 (FHL2,        perforin mutation), Agammaglobulinemia (X-linked        Agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi        syndrome, Hemolytic anemia due to red cell pyruvate kinase        deficiency, Paroxysmal nocturnal hemoglobinuria, X-linked        Adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative        disease, Unicentric Castleman's Disease, Multicentric        Castleman's Disease, Congenital amegakaryocytic thrombocytopenia        (CAMT) type I, Reticular dysgenesis, Fanconi anemia, Acquired        idiopathic sideroblastic anemia, Systemic mastocytosis, Von        willebrand disease (VWD), Congenital dyserythropoietic anemia        type 2, Cartilage-hair hypoplasia syndrome, Hereditary        spherocytosis, Blackfan-Diamond syndrome, Shwachman-Diamond        syndrome, Thrombocytopenia-absent radius syndrome,        Osteopetrosis, Infantile osteopetrosis, Mucopolysaccharidoses,        Lesch-Nyhan syndrome, Glycogen storage disease, Congenital        mastocytosis, Omenn syndrome, X-linked Immunodysregulation,        polyendocrinopathy, and enteropathy (IPEX), IPEX characterized        by mutations in FOXP3, X-linked syndrome of polyendocrinopathy,        immune dysfunction, and diarrhea (XPID), X-Linked        Autoimmunity-Allergic Dysregulation Syndrome (XLAAD), IPEX-like        syndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3,        Hyper IgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin        M, Bare lymphocyte Syndrome type I, and Bare lymphocyte Syndrome        type II (Bare lymphocyte Syndrome type II, MHC class I        deficiency; Bare lymphocyte Syndrome type II, complementation        group A; Bare lymphocyte Syndrome type II, complementation group        C; Bare lymphocyte Syndrome type II complementation group D;        Bare lymphocyte Syndrome type II, complementation group E).        99. The method of any one of paragraphs 80-96, wherein said        recipient is a human patient suffering from a hematolymphoid        malignancy, a non-hematolymphoid malignancy, or a protein        deficiency, or a tissue or cell transplantation recipient (e.g.,        to induce tolerance to transplanted tissue or cells).        100. The method of any one of paragraphs 80-99, wherein said        hematopoietic stem cells are autologous or syngeneic.        101. The method of any one of paragraphs 80-99, wherein said        hematopoietic stem cells are allogeneic.        102. A composition comprising one or more agents that together        exhibit two or more activities selected from the group        consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation.        103. The composition of paragraph 102, wherein the modulation of        histone methylation is activation of histone methylation,        maintenance of histone methylation, or inhibition of histone        demethylation.        104. The composition of paragraph 102 or 103, wherein the        modulation of histone acetylation is activation of histone        acetylation, maintenance of histone acetylation, or inhibition        of histone deacetylation.        105. The composition of paragraph 103, wherein said one or more        agents comprise a compound that activates histone methylation,        maintains histone methylation, or inhibits histone demethylation        and a compound that inhibits TGFβ signaling.        106. The composition of paragraph 105, wherein said compound        that activates histone methylation, maintains histone        methylation, or inhibits histone demethylation a histone        demethylase inhibitor and said compound that inhibits TGFβ        signaling is a TGFβ receptor inhibitor.        107. The composition of paragraph 106, wherein said histone        demethylase inhibitor is a LSD1 inhibitor.        108. The composition of paragraph 107, wherein said LSD1        inhibitor is LSD1 inhibitor IV RN-1 and said TGFβ receptor        inhibitor is ALK5 inhibitor II.        109. The composition of paragraph 107, wherein said LSD1        inhibitor is tranylcypromine and said TGFβ receptor inhibitor is        ALK5 inhibitor II.        110. A composition comprising one or more agents that together        inhibit the activity of two or more proteins selected from the        group consisting of:    -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase.        111. The composition of paragraph 110, wherein said histone        demethylase is LSD1.        112. The composition of paragraph 110, wherein said one or more        agents comprise a histone demethylase inhibitor selected from        the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor        II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007,        LSD1 inhibitor I, and Tranylcypromine.        113. The composition of any one of paragraphs 110-112, wherein        said protein that propagates TGFβ signaling is a TGFβ receptor.        114. The composition of any one of paragraphs 110-113, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates TGFβ signaling selected from the group        consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1.        115. The composition of any one of paragraphs 110-114, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates p38 signaling, and wherein said compound        is SB203580.        116. The composition of any one of paragraphs 110-115, wherein        said one or more agents comprise a compound that inhibits a        protein that promotes β-catenin degradation selected from the        group consisting of CHIR99021, lithium chloride, BIO, and FGF2.        117. The composition of any one of paragraphs 110-116, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase selected from the group consisting of        Trichostatin A, valproic acid, butyrylhydroxamic acid, and        istodax.        118. The composition of any one of paragraphs 110-117, wherein        said one or more agents together inhibit the activity of a        histone demethylase and a protein that propagates TGFβ signaling        119. The composition of paragraph 118, wherein said histone        demethylase is LSD1.        120. The composition of paragraph 118 or 119, wherein said        protein that propagates TGFβ signaling is a TGFβ receptor.        121. The composition of any one of paragraphs 118-120, wherein        said one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5        inhibitor II.        122. The composition of any one of paragraphs 118-121, wherein        said one or more agents comprise a compound that inhibits p38        signaling.        123. The composition of any one of paragraphs 118-122, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase.        124. The composition of any one of paragraphs 118-123, wherein        said one or more agents comprise a compound that inhibits BMP        signaling.        125. A composition comprising (a) a first agent selected from        the group consisting of an LSD1 inhibitor IV RN-1, LSD1        inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III        CBB1007, LSD1 inhibitor I, and Tranylcypromine, and (b) a second        agent selected from the group consisting of ALK5 inhibitor II,        LY364947, A83-01, Trichostatin A, SB203580, CHIR99021, DMH1,        sodium acetate, and istodax.        126. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts that are        sufficient to produce an expanded population of hematopoietic        stem cells.        127. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts that are        sufficient to produce a population of cells enriched with        hematopoietic stem cells.        128. The composition of any one of paragraphs 102-125, wherein        said one or more agents are present in amounts sufficient to        maintain the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells for at least two        days.        129. The composition of any one of paragraphs 126-128, wherein        said one or more agents are present in an aqueous solution.        130. The composition of any one of paragraphs 126-128, wherein        said one or more agents are present as a lyophilized solid.        131. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to stimulate expansion of said population of cells by        10% or more relative to a population of hematopoietic stem cells        that have been contacted with a substance that inhibits aryl        hydrocarbon receptor signaling such as SR1 or an analog thereof,        UM171 or an analog thereof, a UM171 analog selected from Table        11, a prostaglandin, an agonist of Notch signaling, or an        inhibitor of SIRT1 such as nicotinamide, cambinol, or an analog        thereof, after seven or more days of culture (e.g., after seven,        ten, twelve, fourteen, fifteen, twenty, or more days of        culture).        132. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to enrich the population of cells with hematopoietic        stem cells by 10% or more relative to a population of        hematopoietic stem cells that have been contacted with a        substance that inhibits aryl hydrocarbon receptor signaling such        as SR1 or an analog thereof, UM171 or an analog thereof, a UM171        analog selected from Table 11, a prostaglandin, an agonist of        Notch signaling, or an inhibitor of SIRT1 such as nicotinamide,        cambinol, or an analog thereof, after seven or more days of        culture (e.g., after seven, ten, twelve, fourteen, fifteen,        twenty, or more days of culture).        133. The composition of any one of paragraphs 126-130, wherein        said one or more agents are present in amounts that are        sufficient to maintain long term engraftment potential of said        hematopoietic stem cells post-transplantation after having        contacted said cells in culture for two or more days (e.g.,        three, five, seven, ten, twelve, fourteen, fifteen, twenty, or        more days).        134. A composition comprising a combination of agents selected        from the combination of agents of Table 1, Table 2, Table 3,        Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, and Table        10.        135. A cell culture medium comprising the composition of any one        of paragraphs 102-134.        136. The cell culture medium of paragraph 135, wherein said cell        culture medium is substantially free of serum.        137. The composition of any one of paragraphs 102-134, wherein        said composition further comprises a population of hematopoietic        stem cells in contact with said one or more agents.        138. The composition of paragraph 137, wherein said        hematopoietic stem cells have been cultured in the presence of        said one or more agents for two or more days (e.g., three, five,        seven, ten, twelve, fourteen, fifteen, twenty, or more days).        139. A method of producing an expanded population of        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic stem cells with    -   (1) a first agent that exhibits one or more activities selected        from the group consisting of:        -   a. modulation of histone methylation;        -   b. inhibition of TGFβ signaling;        -   c. inhibition of p38 signaling;        -   d. activation of canonical Wnt signaling; and        -   e. modulation of histone acetylation; and    -   (2) a second agent selected from the group consisting of SR1 or        an analog thereof, UM171 or an analog thereof, a UM171 analog        selected from Table 11, a prostaglandin, an agonist of Notch        signaling, or an inhibitor of SIRT1, wherein the first and        second agents are present in amounts that together are        sufficient to produce an expanded population of hematopoietic        stem cells.        140. A method of enriching a population of cells with        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic cells that contains one        or more hematopoietic stem cells with (1) a first agent that        exhibits one or more activities selected from the group        consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling; and    -   e. modulation of histone acetylation; and        (2) a second agent selected from the group consisting of SR1 or        an analog thereof, UM171 or an analog thereof, a UM171 analog        selected from Table 11, a prostaglandin, an agonist of Notch        signaling, or an inhibitor of SIRT1, wherein the first and        second agents are present in amounts that together are        sufficient to produce a population of cells enriched with        hematopoietic stem cells.        141. A method of maintaining the hematopoietic stem cell        functional potential of a population of hematopoietic stem cells        ex vivo for at least two days, said method comprising contacting        a first population of hematopoietic stem cells with    -   (1) a first agent that exhibits one or more activities selected        from the group consisting of:        -   a. modulation of histone methylation;        -   b. inhibition of TGFβ signaling;        -   c. inhibition of p38 signaling;        -   d. activation of canonical Wnt signaling; and        -   e. modulation of histone acetylation; and    -   (2) a second agent selected from the group consisting of SR1 or        an analog thereof, UM171 or an analog thereof, a UM171 analog        selected from Table 11, a prostaglandin, an agonist of Notch        signaling, or an inhibitor of SIRT1, wherein the population of        hematopoietic stem cells exhibits a hematopoietic stem cell        functional potential after two or more days that is greater than        that of a control population of hematopoietic stem cells        cultured under the same conditions and for the same time as said        population of hematopoietic stem cells but not contacted with        said one or more agents and said one or more substances.        142. A population of hematopoietic stem cells produced by the        method of any one of paragraphs 1-101 and 139-141.        143. A kit comprising the composition of any one of paragraphs        102-134, 137, and 138, wherein said kit further comprises a        package insert.        144. The kit of paragraph 143, wherein said package insert        instructs a user of said kit to expand, enrich, or maintain the        hematopoietic stem cell functional potential of a population of        hematopoietic stem cells ex vivo.        145. The kit of paragraph 143, wherein said package insert        instructs said user to express a polynucleotide in said        hematopoietic stem cells.        146. The kit of paragraph 143, wherein said package insert        instructs said user to administer said hematopoietic stem cells        to a patient.        147. A method of producing an expanded population of        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic stem cells with one or        more agents that together exhibit two or more activities        selected from the group consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor signaling;

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

148. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together exhibit two or more activitiesselected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation, and    -   f. inhibition of aryl hydrocarbon receptor signaling;

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

149. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together exhibittwo or more activities selected from the group consisting of:

-   -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation and    -   f. inhibition of aryl hydrocarbon receptor signaling;

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

150. The method of any one of paragraphs 147-149, wherein the modulationof histone methylation is activation of histone methylation, maintenanceof histone methylation, or inhibition of histone demethylation.

151. The method of any one of paragraphs 147-150, wherein the modulationof histone acetylation is activation of histone acetylation, maintenanceof histone acetylation, or inhibition of histone deacetylation.

152. The method of paragraph 151, wherein said one or more agentscomprise a compound that activates histone methylation, maintainshistone methylation, or inhibits histone demethylation and a compoundthat inhibits TGFβ signaling.

153. The method of paragraph 152, wherein said compound that activateshistone methylation, maintains histone methylation, or inhibits histonedemethylation is a histone demethylase inhibitor and said compound thatinhibits TGFβ signaling is a TGFβ receptor inhibitor.154. The method of paragraph 153, wherein said histone demethylaseinhibitor is a LSD1 inhibitor.155. The method of paragraph 154, wherein said LSD1 inhibitor is LSD1inhibitor IV RN-1 and said TGFβ receptor inhibitor is ALK5 inhibitor II.156. The method of paragraph 155, wherein said LSD1 inhibitor istranylcypromine and said TGFβ receptor inhibitor is ALK5 inhibitor II.157. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor;

wherein the one or more agents are present in amounts that aresufficient to produce an expanded population of hematopoietic stemcells.

158. A method of enriching a population of cells with hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic cells that contains one or more hematopoietic stem cellswith one or more agents that together inhibit the activity of two ormore proteins selected from the group consisting of:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor;

wherein the one or more agents are present in amounts that aresufficient to produce a population of cells enriched with hematopoieticstem cells.

159. A method of maintaining the hematopoietic stem cell functionalpotential of a population of hematopoietic stem cells ex vivo for atleast two days, said method comprising contacting a first population ofhematopoietic stem cells with one or more agents that together inhibitthe activity of two or more proteins selected from the group consistingof:

-   -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation;    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor;

wherein the first population of hematopoietic stem cells exhibits ahematopoietic stem cell functional potential after two or more days thatis greater than that of a control population of hematopoietic stem cellscultured under the same conditions and for the same time as said firstpopulation of hematopoietic stem cells but not contacted with said oneor more agents.

160. The method of any one of paragraphs 147-159, wherein the one ormore agents comprise a combination of agents selected from thecombination of agents of Table 1, Table 2, Table 3, Table 4, Table 5,and Table 6.

161. The method of any one of paragraphs 147-160, wherein said histonedemethylase is LSD1.

162. The method of any one of paragraphs 147-161, wherein said one ormore agents comprise a histone demethylase inhibitor selected from thegroup consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I,and Tranylcypromine.163. The method of any one of paragraphs 147-162, wherein said proteinthat propagates TGF signaling is a TGFβ receptor.164. The method of any one of paragraphs 147-163, wherein said one ormore agents comprise a compound that inhibits a protein that propagatesTGFβ signaling selected from the group consisting of ALK5 inhibitor II,LY364947, A83-01, and DMH1.165. The method of any one of paragraphs 147-164, wherein said one ormore agents comprise a compound that inhibits a protein that propagatesp38 signaling, and wherein said compound is SB203580.166. The method of any one of paragraphs 147-165, wherein said one ormore agents comprise a compound that inhibits a protein that promotesβ-catenin degradation selected from the group consisting of CHIR99021,lithium chloride, BIO, and FGF2.167. The method of any one of paragraphs 147-166, wherein said one ormore agents comprise a compound that inhibits a histone deacetylase areselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.168. The method of any one of paragraphs 147-167, wherein said one ormore agents together inhibit the activity of a histone demethylase and aprotein that propagates TGFβ signaling.169. The method of paragraph 168, wherein said histone demethylase isLSD1.170. The method of paragraph 168-169, wherein said protein thatpropagates TGFβ signaling is a TGFβ receptor.171. The method of any one of paragraphs 168-170, wherein said one ormore agents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II.172. The method of any one of paragraphs 147-171, wherein said one ormore agents comprise a compound that inhibits p38 signaling.173. The method of any one of paragraphs 147-172, wherein said one ormore agents comprise a compound that inhibits a histone deacetylase.174. The method of any one of paragraphs 147-173, wherein said one ormore agents further comprise a compound that inhibits BMP signaling.175. The method of any one of paragraphs 147-174, wherein the substancethat inhibits aryl hydrocarbon receptor signaling is SR1 or an analogthereof.176. A method of producing an expanded population of hematopoietic stemcells ex vivo, said method comprising contacting a population ofhematopoietic stem cells with (a) a first agent selected from the groupconsisting of an LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, andTranylcypromine, and (b) a second agent selected from the groupconsisting of ALK5 inhibitor II, LY364947, A83-01, Trichostatin A,SB203580, CHIR99021, DMH1, sodium acetate, and istodax.177. The method of any one of paragraphs 147-176, wherein said one ormore agents are present in amounts that are sufficient to stimulateexpansion of said population of cells by 10% or more relative to apopulation of hematopoietic stem cells not contacted with said one ormore agents after seven or more days of culture (e.g., after seven, ten,twelve, fourteen, fifteen, twenty, or more days of culture).178. The method of any one of paragraphs 147-177, wherein said one ormore agents are present in amounts that are sufficient to stimulateexpansion of said population of cells by 10% or more relative to apopulation of hematopoietic stem cells that have been contacted with asubstance that inhibits aryl hydrocarbon receptor signaling such as SR1or an analog thereof, UM171 or an analog thereof, a UM171 analogselected from Table 11, a prostaglandin, an agonist of Notch signaling,or an inhibitor of SIRT1 such as nicotinamide, cambinol, or an analogthereof, after seven or more days of culture (e.g., after seven, ten,twelve, fourteen, fifteen, twenty, or more days of culture).179. The method of any one of paragraphs 147-178, wherein said one ormore agents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells not contacted withsaid one or more agents after seven or more days of culture (e.g., afterseven, ten, twelve, fourteen, fifteen, twenty, or more days of culture).180. The method of any one of paragraphs 147-179, wherein said one ormore agents are present in amounts that are sufficient to enrich saidpopulation of cells with hematopoietic stem cells by 10% or morerelative to a population of hematopoietic stem cells that have beencontacted with a substance that inhibits aryl hydrocarbon receptorsignaling such as SR1 or an analog thereof, UM171 or an analog thereof,a UM171 analog selected from Table 11, a prostaglandin, an agonist ofNotch signaling, or an inhibitor of SIRT1 such as nicotinamide,cambinol, or an analog thereof, after seven or more days of culture(e.g., after seven, ten, twelve, fourteen, fifteen, twenty, or more daysof culture).181. The method of any one of paragraphs 147-180, wherein said firstpopulation of hematopoietic stem cells exhibits a hematopoietic stemcell functional potential after three or more days of culture (e.g.,three, five, seven, ten, twelve, fourteen, fifteen, twenty, or moredays) that is greater than that of said control population ofhematopoietic stem cells.182. The method of any one of paragraphs 147-181, wherein saidhematopoietic stem cells are mammalian cells.183. The method of paragraph 182, wherein said mammalian cells are humancells.184. The method of paragraph 183, wherein said hematopoietic stem cellsare CD34+ cells.185. The method of paragraph 184, wherein at least 10% of said CD34+cells are CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, orCD34+CD38-CD90+CD45RA-CD49F+ cells.186. The method of any one of paragraphs 183-185, wherein saidhematopoietic stem cells are from human cord blood.187. The method of any one of paragraphs 183-185, wherein saidhematopoietic stem cells are from human mobilized peripheral blood.188. The method of any one of paragraphs 183-185, wherein saidhematopoietic stem cells are from human bone marrow.189. The method of any one of paragraphs 183-188, wherein saidhematopoietic stem cells are freshly isolated from said human.190. The method of any one of paragraphs 183-189, wherein saidhematopoietic stem cells have been previously cryopreserved.191. The method of paragraph 182, wherein said mammalian cells aremurine cells.192. The method of any one of paragraphs 147-191, wherein saidhematopoietic stem cells are cultured for two or more days (e.g., three,five, seven, ten, twelve, fourteen, fifteen, twenty, or more days).193. The method of any one of paragraphs 147-192, wherein saidhematopoietic stem cells contact said one or more agents for two or moredays (e.g., three, five, seven, ten, twelve, fourteen, fifteen, twenty,or more days).194. The method of any one of paragraphs 147-193, wherein saidhematopoietic stem cells are contacted with said one or more agentssimultaneously.195. The method of any one of paragraphs 147-194, wherein saidhematopoietic stem cells are contacted with said one or more agents atdifferent times.196. The method of any one of paragraphs 147-195, wherein saidhematopoietic stem cells maintain hematopoietic stem cell functionalpotential after two days (e.g., three, five, seven, ten, twelve,fourteen, fifteen, twenty, or more days) in culture.197. The method of paragraph 196, wherein said hematopoietic stem cellsmaintain hematopoietic stem cell functional potential followingtransplantation after two days (e.g., three, five, seven, ten, twelve,fourteen, fifteen, twenty, or more days) in culture.198. The method of any one of paragraphs 47-197, wherein saidhematopoietic stem cells maintain long term engraftment potential aftertwo days (e.g., three, five, seven, ten, twelve, fourteen, fifteen,twenty, or more days) in culture.199. The method of any one of paragraphs 147-198, wherein upontransplantation into a patient, said hematopoietic stem cells give riseto recovery of a population of cells selected from the group consistingof neutrophils, platelets, red blood cells, monocytes, macrophages,antigen-presenting cells, microglia, osteoclasts, dendritic cells, andlymphocytes.200. The method of paragraph 199, wherein said lymphocytes are selectedfrom the group consisting of natural killer cells, T cells (e.g., CD4+or CD8+ cells), and B cells.201. The method of any one of paragraphs 147-200, wherein saidhematopoietic stem cells are capable of localizing to hematopoietictissue to reestablish productive hematopoiesis in a transplantedrecipient.202. The method of any one of paragraphs 147-201, wherein saidhematopoietic stem cells are cultured on a plastic surface or on asubstrate that includes vitronectin, fibronectin, or matrigel.203. The method of any one of paragraphs 147-202, wherein saidhematopoietic stem cells are cultured in the presence of 2-20% oxygen.204. The method of paragraph 203, wherein said hematopoietic stem cellsare cultured in the presence of 2-12% oxygen.204. The method of paragraph 203, wherein said hematopoietic stem cellsare cultured in the presence of about 5% oxygen.205. The method of any one of paragraphs 147-204, wherein saidhematopoietic stem cells are originally within a mononuclear cellfraction prior to treatment with said one or more agents.206. The method of any one of paragraphs 147-205, wherein saidhematopoietic stem cells are originally within a CD34+, CD34+CD38-,CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA−, or CD34+CD38-CD90+CD45RA-CD49F+enriched cell fraction prior to contacting said one or more agents.207. The method of any one of paragraphs 147-206, wherein saidhematopoietic stem cells are originally within an un-enriched cellfraction prior to contacting said one or more agents.208. A method of introducing a polynucleotide into a population ofhematopoietic stem cells, said method comprising:

-   -   a. inserting the polynucleotide into said population of        hematopoietic stem cells; and    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 147-207, or        maintaining the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells according to the        method of any one of paragraphs 147-207.        209. The method of paragraph 208, wherein (a) precedes (b).        210. The method of paragraph 208, wherein (b) precedes (a).        211. The method of any one of paragraphs 208-201, wherein said        method comprises providing one or more reagents that cleave a        nucleic acid in said cells.        212. The method of paragraph 211, wherein the one or more        reagents that cleave a nucleic acid in said cells comprise a        zinc finger nuclease.        213. The method of paragraph 211, wherein the one or more        reagents that cleave a nucleic acid in said cells comprise a        transcription activator-like effector nuclease.        214. The method of paragraph 211, wherein the one or more        reagents that cleave a nucleic acid in said cells comprise a        CRISPR-associated protein.        215. The method of paragraph 211, wherein the one or more agents        that cleave a nucleic acid in said cells comprise a        meganuclease.        216. The method of any one of paragraphs 208-215, wherein said        method comprises contacting the hematopoietic stem cells with a        vector selected from the group consisting of a viral vector        (such as retrovirus, adenovirus, parvovirus, coronavirus,        rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpes        virus, or poxvirus) and a transposable element (such as a        piggybac transposon or sleeping beauty transposon)        217. The method of any one of paragraphs 208-216, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by electroporation, Nucleofection™, or        squeeze-poration.        218. The method of any one of paragraphs 208-217, wherein said        method comprises contacting the cells with a transformation        agent selected from the group consisting of a cationic polymer        (e.g., diethylaminoethyl-dextran), a cationic lipid, calcium        phosphate, an activated dendrimer, and a magnetic bead.        219. The method of any one of paragraphs 208-218, wherein said        method comprises introducing said polynucleotide into said        hematopoietic stem cells by microinjection or laserfection.        220. The method of any one of paragraphs 208-219, wherein said        polynucleotide comprises a regulatory sequence selected from the        group consisting of a promoter, enhancer, or silencer sequence.        221. The method of any one of paragraphs 208-220, wherein said        polynucleotide encodes a molecule selected from the group        consisting of a protein and a RNA (mRNA, tRNA, siRNA, miRNA,        shRNA).        222. The method of any one of paragraphs 208-221, wherein said        polynucleotide is a chemically modified RNA.        223. The method of any one of paragraphs 208-222, wherein said        method further comprises introducing the population of expanded        hematopoietic stem cells or progeny thereof into a recipient.        224. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. expanding said population of hematopoietic stem cells        according to the method of any one of paragraphs 147-207;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of expanded hematopoietic stem        cells or progeny thereof into said recipient.        225. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. enriching said population of hematopoietic stem cells        according to the method of any one of paragraphs 147-207;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing the population of cells enriched with        hematopoietic stem cells or progeny thereof into said recipient.        226. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells;    -   b. maintaining the hematopoietic stem cell functional potential        of said population of hematopoietic stem cells according to the        method of any one of paragraphs 147-207;    -   c. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   d. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        227. A method of treating a recipient with hematopoietic stem        cells or progeny thereof, said method comprising:    -   a. providing a population of hematopoietic stem cells produced        by the method of any one of paragraphs 147-207;    -   b. optionally differentiating said hematopoietic stem cells into        common lymphoid progenitor cells, common myeloid progenitor        cells, megakaryocyte-erythroid progenitor cells,        granulocyte-megakaryocyte progenitor cells, granulocytes,        promyelocytes, neutrophils, eosinophils, basophils,        erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,        platelet-producing megakaryocytes, platelets, monocytes,        macrophages, dendritic cells, microglia, osteoclasts, and        lymphocytes, NK cells, B-cells and/or T-cells; and    -   c. introducing said population of hematopoietic stem cells or        progeny thereof into said recipient.        228 The method of any one of paragraphs 224-227, wherein said        recipient is a human.        229. The method of paragraph 228, wherein said hematopoietic        stem cells are derived from one or more hematopoietic stem cells        isolated from a human donor.        230. The method of paragraph 229, wherein said hematopoietic        stem cells are from mobilized peripheral blood of said donor.        231. The method of paragraph 228-229, wherein said donor has        been previously administered one or more mobilizing agents        selected from the group consisting of a CXCR4 antagonist (e.g.,        AMD3100), GCSF, and GROβ.        232. The methods of any one of paragraphs 147-231, wherein the        hematopoietic stem cells are additionally contacted with UM171,        an analog thereof, or a UM171 analog selected from Table 11.        233. The methods of any one of paragraphs 147-232, wherein the        hematopoietic stem cells are additionally contacted with a        compound selected from Table 11.        234. The method of any one of paragraphs 147-233, wherein the        hematopoietic stem cells are additionally contacted with a        prostaglandin        235. The method of paragraph 234, wherein said prostaglandin is        dmPGE2 or an analog thereof.        236. The method of any one of paragraphs 147-235, wherein the        hematopoietic stem cells are additionally contacted with an        agonist of Notch signaling.        237. The method of any one of paragraphs 147-236, wherein the        hematopoietic stem cells are additionally contacted with an        inhibitor of SIRT1.        238. The method of paragraph 237, wherein said inhibitor or        SIRT1 is selected from the group consisting of nicotinamide,        cambinol, and analogs thereof.        239. The method of any one of paragraphs 224-238, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Acute Lymphoblastic Leukemia (ALL),        Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia        (CML), Chronic Lymphocytic Leukemia (CLL), Hodgkin Lymphoma        (HL), Non-Hodgkin Lymphoma (NHL), Myelodysplastic Syndrome        (MDS), Multiple myeloma, Aplastic anemia, Bone marrow failure,        Myeloproliferative disorders such as Myelofibrosis, Essential        thrombocytopenia or Polycythemia vera, Fanconi anemia,        Dyskeratosis congenita, Common variable immune deficiency (CVID,        such as CVID 1, CVID 2, CVID 3, CVID 4, CVID 5, and CVID 6),        Human immunodeficiency virus (HIV), Hemophagocytic        lymphohistiocystosis, Amyloidosis, Solid tumors such as        Neuroblastoma, Germ cell tumors, Breast cancer, Wilms' tumor,        Medulloblastoma, and Neuroectodermal tumors, Autoimmune diseases        such as Scleroderma, Multiple sclerosis, Ulcerative colitis,        Systemic lupus erythematosus and Type I diabetes, or protein        deficiencies such as Adrenoleukodystrophy (ALD), Metachromatic        leukodystrophy (MLD), Hemophilia A & B, Hurler syndrome, Hunter        syndrome, Fabry disease, Gaucher disease, Epidermolysis bullosa,        Globoid Cell Leukodystrophy, Sanfillipo syndrome, and Morquio        syndrome.        240. The method of any one of paragraphs 224-238, wherein said        recipient is a human patient suffering from a disease selected        from the group consisting of Sickle cell anemia, Alpha        thalassemia, Beta thalassemia, Delta thalassemia, Hemoglobin        E/thalassemia, Hemoglobin S/thalassemia, Hemoglobin        C/thalassemia, Hemoglobin D/thalassemia, Chronic granulomatous        disease (X-linked Chronic granulomatous disease, autosomal        recessive (AR) chronic granulomatous disease, chronic        granulomatous disease AR I NCF1, Chronic granulomatous disease        AR CYBA, Chronic granulomatous disease AR II NCF2, Chronic        granulomatous disease AR III NCF4), X-linked Severe Combined        Immune Deficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID,        Rag1/Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, Congenital        agranulocytosis, Congenital agranulocytosis-congenital        neutropenia-SCN1, Congenital agranulocytosis-congenital        neutropenia-SCN2, Familial hemophagocytic lymphohistiocystosis        (FHL), Familial hemophagocytic lymphohistiocytosis type 2 (FHL2,        perforin mutation), Agammaglobulinemia (X-linked        Agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi        syndrome, Hemolytic anemia due to red cell pyruvate kinase        deficiency, Paroxysmal nocturnal hemoglobinuria, X-linked        Adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative        disease, Unicentric Castleman's Disease, Multicentric        Castleman's Disease, Congenital amegakaryocytic thrombocytopenia        (CAMT) type I, Reticular dysgenesis, Fanconi anemia, Acquired        idiopathic sideroblastic anemia, Systemic mastocytosis, Von        willebrand disease (VWD), Congenital dyserythropoietic anemia        type 2, Cartilage-hair hypoplasia syndrome, Hereditary        spherocytosis, Blackfan-Diamond syndrome, Shwachman-Diamond        syndrome, Thrombocytopenia-absent radius syndrome,        Osteopetrosis, Infantile osteopetrosis, Mucopolysaccharidoses,        Lesch-Nyhan syndrome, Glycogen storage disease, Congenital        mastocytosis, Omenn syndrome, X-linked Immunodysregulation,        polyendocrinopathy, and enteropathy (IPEX), IPEX characterized        by mutations in FOXP3, X-linked syndrome of polyendocrinopathy,        immune dysfunction, and diarrhea (XPID), X-Linked        Autoimmunity-Allergic Dysregulation Syndrome (XLAAD), IPEX-like        syndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3,        Hyper IgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin        M, Bare lymphocyte Syndrome type I, and Bare lymphocyte Syndrome        type II (Bare lymphocyte Syndrome type II, MHC class I        deficiency; Bare lymphocyte Syndrome type II, complementation        group A; Bare lymphocyte Syndrome type II, complementation group        C; Bare lymphocyte Syndrome type II complementation group D;        Bare lymphocyte Syndrome type II, complementation group E).        241. The method of any one of paragraphs 224-238, wherein said        recipient is a human patient suffering from a hematolymphoid        malignancy, a non-hematolymphoid malignancy, or a protein        deficiency, or a tissue or cell transplantation recipient (e.g.,        to induce tolerance to transplanted tissue or cells).        242. The method of any one of paragraphs 224-241, wherein said        hematopoietic stem cells are autologous or syngeneic.        243. The method of any one of paragraphs 224-241, wherein said        hematopoietic stem cells are allogeneic.        244. A composition comprising one or more agents that together        exhibit two or more activities selected from the group        consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor.        245. The composition of paragraph 244, wherein the modulation of        histone methylation is activation of histone methylation,        maintenance of histone methylation, or inhibition of histone        demethylation.        246. The composition of paragraph 244 or 245, wherein the        modulation of histone acetylation is activation of histone        acetylation, maintenance of histone acetylation, or inhibition        of histone deacetylation.        247. The composition of any of paragraphs 244-246, wherein said        one or more agents comprise a compound that activates histone        methylation, maintains histone methylation, or inhibits histone        demethylation and a compound that inhibits TGFβ signaling.        248. The composition of any of paragraphs 244-247, wherein said        compound that activates histone methylation, maintains histone        methylation, or inhibits histone demethylation a histone        demethylase inhibitor and said compound that inhibits TGFβ        signaling is a TGFβ receptor inhibitor.        249. The composition of any of paragraphs 244-248, wherein said        histone demethylase inhibitor is a LSD1 inhibitor.        250. The composition of paragraph 249, wherein said LSD1        inhibitor is LSD1 inhibitor IV RN-1 and said TGFβ receptor        inhibitor is ALK5 inhibitor II.        251. The composition of any of paragraphs 244-250, wherein said        LSD1 inhibitor is tranylcypromine and said TGFβ receptor        inhibitor is ALK5 inhibitor II.        252. A composition comprising one or more agents that together        inhibit the activity of two or more proteins selected from the        group consisting of:    -   a. a histone demethylase;    -   b. a protein that propagates TGFβ signaling;    -   c. a protein that propagates p38 signaling;    -   d. a protein that promotes β-catenin degradation; and    -   e. a histone deacetylase; and    -   f. aryl hydrocarbon receptor.        253. The composition of paragraph 252, wherein said histone        demethylase is LSD1.        254. The composition of paragraph 252, wherein said one or more        agents comprise a histone demethylase inhibitor selected from        the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor        II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007,        LSD1 inhibitor I, and Tranylcypromine.        255. The composition of any one of paragraphs 252-254, wherein        said protein that propagates TGFβ signaling is a TGFβ receptor.        256. The composition of any one of paragraphs 252-255, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates TGFβ signaling selected from the group        consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1.        257. The composition of any one of paragraphs 252-256, wherein        said one or more agents comprise a compound that inhibits a        protein that propagates p38 signaling, and wherein said compound        is SB203580.        258. The composition of any one of paragraphs 252-257, wherein        said one or more agents comprise a compound that inhibits a        protein that promotes β-catenin degradation selected from the        group consisting of CHIR99021, lithium chloride, BIO, and FGF2.        259. The composition of any one of paragraphs 252-258, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase selected from the group consisting of        Trichostatin A, valproic acid, butyrylhydroxamic acid, and        istodax.        260. The composition of any one of paragraphs 252-259, wherein        said one or more agents together inhibit the activity of a        histone demethylase and a protein that propagates TGFβ signaling        261. The composition of any of paragraphs 252-260, wherein said        histone demethylase is LSD1.        262. The composition of paragraph 260 or 261, wherein said        protein that propagates TGFβ signaling is a TGFβ receptor.        263. The composition of any one of paragraphs 260-262, wherein        said one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5        inhibitor II.        264. The composition of any one of paragraphs 260-263, wherein        said one or more agents comprise a compound that inhibits p38        signaling.        265. The composition of any one of paragraphs 260-264, wherein        said one or more agents comprise a compound that inhibits a        histone deacetylase.        266. The composition of any one of paragraphs 260-265, wherein        said one or more agents comprise a compound that inhibits BMP        signaling.        267. The composition of any of paragraphs 244-266, wherein said        one or more agents that inhibit aryl hydrocarbon receptor        signaling comprise SR1.        268. The composition of any one of paragraphs 244-267, wherein        said one or more agents are present in amounts that are        sufficient to produce an expanded population of hematopoietic        stem cells.        269. The composition of any one of paragraphs 244-268, wherein        said one or more agents are present in amounts that are        sufficient to produce a population of cells enriched with        hematopoietic stem cells.        270. The composition of any one of paragraphs 244-269, wherein        said one or more agents are present in amounts sufficient to        maintain the hematopoietic stem cell functional potential of        said population of hematopoietic stem cells for at least two        days.        271. The composition of any one of paragraphs 244-270, wherein        said one or more agents are present in an aqueous solution.        272. The composition of any one of paragraphs 244-271, wherein        said one or more agents are present as a lyophilized solid.        273. The composition of any one of paragraphs 244-272, wherein        said one or more agents are present in amounts that are        sufficient to stimulate expansion of said population of cells by        10% or more relative to a population of hematopoietic stem cells        that have been contacted with a substance that inhibits aryl        hydrocarbon receptor signaling such as SR1 or an analog thereof,        UM171 or an analog thereof, a UM171 analog selected from Table        11, a prostaglandin, an agonist of Notch signaling, or an        inhibitor of SIRT1 such as nicotinamide, cambinol, or an analog        thereof, after seven or more days of culture (e.g., after seven,        ten, twelve, fourteen, fifteen, twenty, or more days of        culture).        274. The composition of any one of paragraphs 244-273, wherein        said one or more agents are present in amounts that are        sufficient to enrich the population of cells with hematopoietic        stem cells by 10% or more relative to a population of        hematopoietic stem cells that have been contacted with a        substance that inhibits aryl hydrocarbon receptor signaling such        as SR1 or an analog thereof, UM171 or an analog thereof, a UM171        analog selected from Table 11, a prostaglandin, an agonist of        Notch signaling, or an inhibitor of SIRT1 such as nicotinamide,        cambinol, or an analog thereof, after seven or more days of        culture (e.g., after seven, ten, twelve, fourteen, fifteen,        twenty, or more days of culture).        275. The composition of any one of paragraphs 244-274, wherein        said one or more agents are present in amounts that are        sufficient to maintain long term engraftment potential of said        hematopoietic stem cells post-transplantation after having        contacted said cells in culture for two or more days (e.g.,        three, five, seven, ten, twelve, fourteen, fifteen, twenty, or        more days).        275. A composition comprising a combination of agents selected        from the combination of agents of Table 1, Table 2, Table 3,        Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, and Table        10.        276. A cell culture medium comprising the composition of any one        of paragraphs 244-275.        277. The cell culture medium of paragraph 276, wherein said cell        culture medium is substantially free of serum.        278. The composition of any one of paragraphs 244-277, wherein        said composition further comprises a population of hematopoietic        stem cells in contact with said one or more agents.        279. The composition of paragraph 278, wherein said        hematopoietic stem cells have been cultured in the presence of        said one or more agents for two or more days (e.g., three, five,        seven, ten, twelve, fourteen, fifteen, twenty, or more days).        280. A method of producing an expanded population of        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic stem cells with    -   (1) a first agent that exhibits one or more activities selected        from the group consisting of:        -   a. modulation of histone methylation;        -   b. inhibition of TGFβ signaling;        -   c. inhibition of p38 signaling;        -   d. activation of canonical Wnt signaling;        -   e. modulation of histone acetylation; and        -   f. inhibition of aryl hydrocarbon receptor signaling; and    -   (2) a second agent selected from the group consisting of UM171        or an analog thereof, a UM171 analog selected from Table 11, a        prostaglandin, an agonist of Notch signaling, or an inhibitor of        SIRT1, wherein the first and second agents are present in        amounts that together are sufficient to produce an expanded        population of hematopoietic stem cells.        281. A method of enriching a population of cells with        hematopoietic stem cells ex vivo, said method comprising        contacting a population of hematopoietic cells that contains one        or more hematopoietic stem cells with (1) a first agent that        exhibits one or more activities selected from the group        consisting of:    -   a. modulation of histone methylation;    -   b. inhibition of TGFβ signaling;    -   c. inhibition of p38 signaling;    -   d. activation of canonical Wnt signaling;    -   e. modulation of histone acetylation; and    -   f. inhibition of aryl hydrocarbon receptor signaling; and        (2) a second agent selected from the group consisting of UM171        or an analog thereof, a UM171 analog selected from Table 11, a        prostaglandin, an agonist of Notch signaling, or an inhibitor of        SIRT1, wherein the first and second agents are present in        amounts that together are sufficient to produce a population of        cells enriched with hematopoietic stem cells.        282. A method of maintaining the hematopoietic stem cell        functional potential of a population of hematopoietic stem cells        ex vivo for at least two days, said method comprising contacting        a first population of hematopoietic stem cells with    -   (1) a first agent that exhibits one or more activities selected        from the group consisting of:        -   a. modulation of histone methylation;        -   b. inhibition of TGFβ signaling;        -   c. inhibition of p38 signaling;        -   d. activation of canonical Wnt signaling;        -   e. modulation of histone acetylation; and        -   f. inhibition of aryl hydrocarbon receptor signaling; and    -   (2) a second agent selected from the group consisting of UM171        or an analog thereof, a UM171 analog selected from Table 11, a        prostaglandin, an agonist of Notch signaling, or an inhibitor of        SIRT1, wherein the population of hematopoietic stem cells        exhibits a hematopoietic stem cell functional potential after        two or more days that is greater than that of a control        population of hematopoietic stem cells cultured under the same        conditions and for the same time as said population of        hematopoietic stem cells but not contacted with said one or more        agents and said one or more substances.        283. A population of hematopoietic stem cells produced by the        method of any one of paragraphs 147-243 and 280-282.        284. A kit comprising the composition of any one of paragraphs        244-279, wherein said kit further comprises a package insert.        285. The kit of paragraph 284, wherein said package insert        instructs a user of said kit to expand, enrich, or maintain the        hematopoietic stem cell functional potential of a population of        hematopoietic stem cells ex vivo.        286. The kit of paragraph 284, wherein said package insert        instructs said user to express a polynucleotide in said        hematopoietic stem cells.        287. The kit of paragraph 284, wherein said package insert        instructs said user to administer said hematopoietic stem cells        to a patient.

EXAMPLES Example 1. Small Molecule Screens to Identify Combinations ofCompounds that Expand, Enrich, and Maintain Cells with HematopoieticStem Cell Properties Upon Ex Vivo Culturing

FIG. 1 shows that HSC potential is rapidly lost upon culture. Peripheralbleed analysis following transplantation into lethally irradiated hostsof freshly isolated murine HSCs (Blue line, circles) or HSCs culturedfor 12 days ex vivo in S-clone+IL12/SCF/TPO+0.75% BSA (red line,squares). Various small molecule screens were conducted in order todetermine optimal combinations of compounds that can be used to expand,enrich, and maintain the hematopoietic stem cell functional potential ofhematopoietic stem cells. FIG. 4 shows a schematic representation of asmall molecule screen for compounds that support HSC ex vivo maintenanceand expansion. 1) Zs·Gr HSC reporter mouse marrow used to isolate HSCs(described in FIGS. 2 and 3A-3C). 2) Isolation of HSCs marked by ZsGrreporter. 3) Development of assay where hit is defined by themaintenance of HSC reporter expression above DMSO control following 6days ex vivo culture. 4) Functional validation of hits in vitro and invivo. FIGS. 5A-5B show the development of a sensitivity assay. FIG. 5A:A total of 200 ZsGr+ and ZsGr−HSCs were seeded/well in various ratios(1:0; 1:1; 1:10; 1:20, 1:100, 0:1—shown as percentage ZsGr+) and imagedusing the Operetta (Perkin Elmer) following 2 days ex vivo culture withindividual cells plotted as being above or below the threshold ofZsGreen detection. FIG. 5B: After 2 days of culture, the percentage ofZsGreen+ cells was determined. This established a minimum number ofcells needed/well to allow for robust detection of ZsGr+ signal after 2days of culturing. FIG. 6 shows a breakdown of pathways targeted inprimary small molecule screens. FIGS. 7A-7B show initial screen resultsfor various library screens. FIG. 7A: Number of compounds screened,initial hits (showing dose response), and validated hits (by flowcytometry to quantify ZSGr+) from each of 4 different libraries of smallmolecules targeting kinases, epigenetic regulators, and G-proteincoupled receptors (GPCR), as well as a peptide library of growthfactors. FIG. 7B: Representative results from 6-point dose response (10uM, 5 uM, 1 uM, 0.5 uM, 0.1 uM, 0.05 uM). * indicates a hit. (See alsoFIGS. 16A-16B).

Example 2. Inhibition of Histone Demethylation and TGFβ Signaling DuringEx Vivo Culturing of HSCs Promotes Expansion, Enrichment, andMaintenance Cells with Hematopoietic Stem Cell Properties

An optimal combination of compounds that can be used to expand, enrich,and maintain the hematopoietic stem cell functional potential ofhematopoietic stem cells includes a histone demethylase inhibitor and aninhibitor of the TGFβ receptor Alk5. FIG. 8 shows the ex vivo culture of20 murine HSCs (LSK34-Flk2-150+ZsGr+) for 7 or 14 days in the presenceof DMSO, LSD1 inhibitor IV (LS), Tgfbeta inhibitor (RepSox) or thecombination of both (C2). Images taken at 4× magnification. Note thatthe cultures in the presence of C2 or LS are more homogenous and lessdifferentiated. FIGS. 9A-9B show the expansion of phenotypic HSCs for 14days ex vivo. 20 murine HSCs (LSK34-Flk2-150+ZsGr+) were cultured in thepresence of DMSO, LSD1 inhibitor IV (LS), Tgfbeta inhibitor RepSox (RS),and the combination of both (C2) for 14 days. This method supportsmaintenance and expansion of ZsGr+ HSCs. FIG. 9A: Representative FACSplots of HSCs cultured for 14 days ex vivo in the presence of DMSO andthe combination of LSD1 inhibitor (LS), Tgfbeta inhibitor RepSox (RS)(C2) showing increased levels of phenotypic HSCs in the presence of C2.FIG. 9B: Number of ZsGr+ HSCs in each condition after 14 days of ex vivoculture. FIGS. 10A-10C show that LSD1 inhibitor (LS), Tgfbeta inhibitorRepSox (RS), and the combination of both (C2) supports maintenance andexpansion of ZsGr+ HSCs. A) Bright-field and ZsGr images of 20 HSCscultured for 4.5 days ex vivo in the presence of LSD1 inhibitor IV (LS),Tgfbeta inhibitor RepSox (RS), and the combination of both (C2). FIG.10B: Frequency of ZsGr+ cells remaining following 4.5 days of ex vivoculture. FIG. 10C: Number of ZsGr+(Green) and ZsGr− (Black) cells within4.5 day cultures (See also FIGS. 11A-13F and 28A-32B).

Example 3. Ex Vivo Culturing HSCs in the Presence of HDAC InhibitorsPromotes Expansion, Enrichment, and Maintenance of Cells withHematopoietic Stem Cell Properties

In addition to inhibitors of histone demethylation and TGFβ signaling,histone deacetylase inhibitors also promote the expansion, enrichment,and maintenance of hematopoietic stem cells ex vivo. FIGS. 22A-22B showsthat structurally distinct HDAC inhibitors function equivalently tomaintain immunophenotypic HSCs. 100 murine HSCs were cultured in thepresence of cytokines only (SCF, TPO, and IL12) in the absence ofcompounds (Standard), or additionally supplemented with a cocktail ofcompounds (Lithium chloride, nicotinamide, N-acetylcysteine, ascorbicacid, A83-01, and SB203580) plus either valproic acid (VPA) ortrichostatin A (TSA), which are structurally distinct HDAC inhibitors.FIG. 22A: Day 7 flow cytometric analysis and FIG. 22B: proportion ofFgd5-ZsGreen+Sca1+ cells for each replicate. FIGS. 41A-41C show thatRomidepsin, a HDAC1/2 specific inhibitor, can replace Trichostatin A, apan HDAC inhibitor, for efficient ex vivo maintenance/expansion of humanHSCs. 3000 CD34+ enriched mobilized peripheral blood cells were culturedfor 7 days in serum free media supplemented with cytokines (SCF, TPO,FLT3L, IL3) in the presence of the indicated chemical combinations(Tgfbeta inhibitor (A, A83-01), pan-HDAC inhibitor (TSA, TrichostatinA), LSD1 inhibitor (TC, Tranylcypromine), p38 inhibitor (p38i,SB203580), HDAC1/2 inhibitor (Rom, Romidepsin)) and analyzed by flowcytometry. FIG. 41A: immunophenotype of the cells, FIG. 41B: percentageof indicated populations, and FIG. 41C: quantification ofimmunophenotypic HSCs (Lineage−CD34+CD45RA-CD38-CD90+CD49F+) 7 dayspost-culturing in the indicated conditions.

Example 4. Secondary Screens to Identify Compounds that PromoteExpansion, Enrichment, and Maintenance of Cells with Hematopoietic StemCell Properties Ex Vivo

A variety of secondary screens were conducted in order to identifyadditional compounds that can be used to expand, enrich, and maintainthe hematopoietic stem cell functional potential of hematopoietic stemcells. FIGS. 14A-14B show secondary small molecule screen for compoundsthat synergize with C2 (LSD1 inhibitor IV and Tgfbeta inhibitor) tosupport HSC ex vivo maintenance and expansion. Schematic of FIG. 14A:primary and FIG. 14B: secondary screen in which 124 potential hitcompounds identified in primary screen were rescreened in the presenceof C2. FIGS. 15A-15C show potential hits of secondary screen that targetpathways of interest including Tgfbeta, histone methylation, histoneacetylation, p38 signaling and Wnt signaling. FIG. 15A: Overview of twostrategies used to identify hits in secondary small molecule screen.FIG. 15B: Hits found by following a strategy based on ZsGreen+ HSCpercentage (strategy 1). FIG. 15C: Hits found by following a strategybased on number of ZsGreen+ HSCs (strategy 2). FIG. 17 shows the resultsof experiments testing compounds reported to maintain murine HSCs:Culturing ZsGreen positive HSCs for 6 days in the presence of; dmPGE2(North, Zon, Nature. 2007), BIO (Ko et al, Stem Cells. 2011), p38inhibitor (Wang et al, Stem Cells Dev. 2011), DMSO: negative control. D2is ZsGr+ HSCs maintained for 2 days ex vivo. Threshold of cellsidentified as ZsGreen+(i.e., HSCs) is shown with arrow. FIGS. 18A-18Bshow a hypothesis driven strategy for modulating candidate pathwaystowards HSC maintenance/expansion. FIG. 18A: Selection of candidatetarget pathways via comparison of intestinal stem cell and hematopoieticstem cell maintenance/proliferation signals. FIG. 18B: Selection ofagents/pathway modulators. FIG. 19 shows a schematic for assessing theactivity of pathway modulators on HSC maintenance/expansion.Fgd5-ZsGreen+immunophenotypic HSCs(Lineage-cKit+Sca1+CD150+CD48-Fgd5-ZsGreen+) were sorted and cultured inthe presence of cytokines only (Standard media) or additionallysupplemented with 7 candidate pathway modulators (W7 media).Multiparametric analysis of cellular immunophenotype was performed byflow cytometry after 14 days of culture. FIG. 20 shows the combinatorialmodulation of 7 candidate pathways maintains and expandsimmunophenotypic HSCs. 50 mouse HSCs were cultured in a serum free mediasupplemented with SCF, TPO, and IL12, plus or minus the seven candidatepathway modulators. Flow cytometric analysis was performed on Day 15.For a description of an example sorting strategy used for flow cytometryanalyses of human cord blood, see FIG. 27.

Example 5. In Vivo Function of Ex Vivo Cultured HSCs

A series of experiments was conducted in order to probe the activity ofex vivo cultured hematopoietic stem cells in in vivo transplantationassays. FIG. 24 shows the in vivo function of murine HSCs cultured for14 days in the presence DMSO (Standard), or compounds targeting 7pathways (Combination) (Tgfbeta inhibitor A83-01, Lsd1 inhibitorTranylcypromine, HDAC inhibitor Trichostatin A, the p38 kinase inhibitorSB203580, BMP inhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodiumacetate). 10 HSCs were cultured for 14 days in the indicated conditionsfollowed by in vivo competitive transplantation into lethally irradiatedhosts (against 2×10⁵ congenically marked bone marrow cells). Peripheralblood donor chimerism at indicated time points post-transplantation areshown. FIGS. 25A-25C show the in vivo function of murine HSCs culturedfor 14 days in the presence DMSO (S-Standard), or compounds targeting 7pathways (W) (Tgfbeta inhibitor A83-01, Lsd1 inhibitor Tranylcypromine,HDAC inhibitor Trichostatin A, the p38 kinase inhibitor SB203580, BMPinhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodium acetate). 100HSCs were cultured for 14 days in the indicated conditions followed byin vivo competitive transplantation (against 2×10⁵ congenically markedbone marrow cells). FIG. 25A: Peripheral blood and FIG. 25B: granulocytedonor chimerism at indicated time points post-transplantation are shown.FIG. 25C: Donor HSC chimerism in the bone marrow of transplantrecipients transplanted with HSCs cultured for 14 days in the indicatedconditions is shown.

Example 6. Relative Contributions of Compounds to HSC Expansion,Maintenance and Enrichment

A series of experiments was conducted in order to determine the relativecontribution of compounds that modulate particular pathways to theexpansion of hematopoietic stem cells. FIGS. 21A-21B show thecontribution of each compound/pathway in ability to maintain and expandphenotypic HSCs during ex vivo culture. 50 murine HSCs (lineage-, ckit+,Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured in the presence ofcytokines only (SCF, TPO, and IL12) in the absence of compounds(standard), or with all 7 compounds (W7), or with subtraction of eachindividual compound (A83-01 (A), Tranylcypromine (TC), Trichostatin A(TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH), Sodium acetate(OAC)). Flow cytometry analysis was performed on Day 14 showing FIG.21A: differentiation to lineage+ cells (stained by antibody cocktailagainst antigens for: B-cells, T-cells, myeloid cells, erythrocyte,granulocyte), where lineage positive is to the right of the dashed line.FIG. 21B: Absolute HSC numbers after 14 days culture in the indicatedconditions from 50 starting HSCs. FIG. 23 shows that the supplementationwith additional compounds reduces heterogeneity of Fgd5+ cells withrespect to CD48 and Sca1 expression. 40 murine HSCs were cultured for 12days in the presence of cytokines (SCF, TPO, and IL12)) and a cocktailof compounds (Lithium chloride, nicotinamide, N-acetylcysteine, ascorbicacid, A83-01, and SB203580, trichostatin A) plus either or both DNAmethyltransferase inhibitor (RG108) and G9a inhibitor (UNC0638). Flowcytometry plots of Fgd5+Lineage− cells from the indicated cultureconditions are shown. The histogram shows the proportions of theindicated subpopulations. FIGS. 35A-35B show that supplementation ofminimal chemical combination with p38 inhibitor improves the yield ofhuman cord blood HSCs. 200 cord blood HSCs were cultured for 12 days inserum free media supplemented with cytokines (SCF, TPO, FLT3L, IL3) inthe presence of the indicated chemical combinations (Tgfbeta inhibitor(A, A83-01), HDAC inhibitor (TSA, Trichostatin A), and LSD1 inhibitor(TC, Tranylcypromine)) or additionally supplemented with p38 inhibitor(p38i, SB203580) and analyzed by flow cytometry. FIG. 35A:Immunophenotype of the cells post-culturing, and FIG. 35B:quantification of immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38-CD90+CD49F+) cultured in the indicatedconditions.

Example 7. Modulation of Multiple Pathways During Ex Vivo CulturingPromotes HSC Expansion, Enrichment, and Maintenance of Cells withHematopoietic Stem Cell Properties

In addition to histone demethylation, TGFβ signaling, and histonedeacetylation, modulation of other pathways additionally promotes HSCexpansion, enrichment, and maintenance of hematopoietic stem cellfunctional potential. FIG. 26 shows that the modulation of four pathwaysis sufficient to maintain/expand immunophenotypic murine HSCs. 50 HSCswere cultured for 14 days in serum free media supplemented withcytokines in the presence of DMSO, or compounds targeting 4 pathways(W4) (Tgfbeta inhibitor A83-01, Lsd1 inhibitor Tranylcypromine, HDACinhibitor Trichostatin A, and the p38 kinase inhibitor SB203580)identified from the initial set of 7 compounds (Tgfbeta inhibitorA83-01, Lsd1 inhibitor Tranylcypromine, HDAC inhibitor Trichostatin A,the p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3betainhibitor Chir99021, and sodium acetate). Immunophenotypic HSCs(Lineage−cKit+Sca1+CD48-CD150+Fgd5ZsGreen+CD41-) were analyzed by flowcytometry. FIGS. 33A-33B show that compounds targeting 7 pathwaysidentified in murine system enable maintenance/expansion ofimmunophenotypic cord blood HSCs. 200 cord blood HSCs were cultured for12 days in serum free media supplemented with cytokines (SCF, TPO,FLT3L, IL3) in the presence of DMSO, or compounds targeting 7 pathways(Combination: Tgfbeta inhibitor A83-01, Lsd1 inhibitor Tranylcypromine,HDAC inhibitor Trichostatin A, the p38 kinase inhibitor SB203580, BMPinhibitor DMH1, Gsk3beta inhibitor Chir99021, and sodium acetate)showing FIG. 33A: Immunophenotype of the cells post-culturing analyzedby flow cytometry, and FIG. 33B: quantification of immunophenotypic HSCs(Lineage−CD34+CD45RA-CD38-CD90+). See also FIGS. 34A-34B, 37A-37C, and40A-40C.

FIGS. 38A-38C show the ex vivo maintenance/proliferation of humanmobilized peripheral blood CD34+ cells using compounds identified usingmurine cells. 3000 CD34+ enriched mobilized peripheral blood cells werecultured for 7 days in serum free media supplemented with cytokines(SCF, TPO, FLT3L, IL3) in the presence of the indicated individualchemicals or chemical combinations (Tgfbeta inhibitor (A, A83-01), HDACinhibitor (TSA, Trichostatin A), LSD1 inhibitor (TC, Tranylcypromine),and p38 inhibitor (p38i, SB203580)) and analyzed by flow cytometry. FIG.38A: Immunophenotype of the cells post-culturing, FIG. 38B: percentageof indicated populations, and FIG. 38C: quantification ofimmunophenotypic HSCs (Lineage−CD34+CD45RA-CD38-CD90+CD49F+)post-culturing in the indicated conditions for 7 days. (W7: A83-01 (A),Tranylcypromine, (TC) Trichostatin A (TSA), SB203580 (p38i), CHIR99021(Chir), DMH1 (DMH), Sodium acetate (OAC), and W3: A83-01 (A),Tranylcypromine, (TC) Trichostatin A (TSA)) FIG. 39 shows that the exvivo culture of human mobilized peripheral blood CD34+ cells usingchemical combination enriches immunophenotypic HSCs. 3000 CD34+ enrichedmobilized peripheral blood cells were cultured for 7 days in serum freemedia supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presenceof the indicated individual chemicals (StemRegenin (SR1), UM171) orcombination of four compounds (W4: Tgfbeta inhibitor (A83-01), HDACinhibitor (Trichostatin A), LSD1 inhibitor (Tranylcypromine), and p38inhibitor (SB203580)) and analyzed by flow cytometry. Quantification ofthe fraction of immunophenotypic HSCs (Lineage−CD34+CD45RA-CD38-CD90+)in CD34+ enriched mobilized peripheral blood prior to ex vivo culture(Uncultured) and post-culturing in the indicated conditions for 7 days.

Example 8. Culturing HSCs Under Low Ambient Oxygen

Culturing hematopoietic stem cells under conditions of reduced oxygenrelative to physiologic levels can be advantageous for hematopoieticstem cell expansion. FIG. 36 shows the cultivation under low oxygentension improves the yield of human cord blood HSCs. 200 cord blood HSCswere cultured in serum free media supplemented with cytokines (SCF, TPO,FLT3L, IL3) and compounds targeting 3 pathways (W3: Tgfbeta inhibitor(A83-01), HDAC inhibitor (Trichostatin A), and LSD1 inhibitor(Tranylcypromine)) for 12 days in either standard tissue cultureincubator (atmospheric oxygen, 21% 02) or low oxygen incubator (5% 02)Immunophenotypic HSCs (Lineage−CD34+CD45RA-CD38-CD90+) cultured in theindicated conditions were quantified post-culturing.

Example 9. Transplanted HSCs

A series of experiments was conducted in order to probe thefunctionality of ex vivo cultured hematopoietic stem cellspost-transplantation. FIGS. 42A-42C show the results of experiments inwhich transplanted human CD34+ cord blood cultured for 14 days ex vivo:The cultures of 10,000 starting CD34+ cord blood cells were transplantedinto sublethally irradiated immunocompromised NSG (Nod-Scid-gamma) micefollowing 14 days of ex vivo culture in the presence of DMSO, W3(Tgfbeta inhibitor A83-01, LSD1 inhibitor tranylcypromine, HDACinhibitor trichostatin A), the combination of LSD1 inhibitor IV and theTgfbeta inhibitor RepSox (C2), Stem Regenin 1 (SR1) and UM171; or 10,000uncultured CD34+ cord blood cells (Fresh) showing FIG. 42A: Peripheralblood donor chimerism. FIG. 42B: Quantification of peripheral blooddonor chimerism at weeks 24 and 30 post transplant, and FIG. 42C:Lineage contribution of transplanted cells at week 30 post-transplant.

Example 10. Transplanted HSCs

A series of experiments was conducted to assess the functionality of exvivo cultured hematopoietic stem cells post-transplantation. FIG. 43depicts the experimental procedure. FIGS. 44-47B depict thecharacterization of the cells after 12 days of culturing in the presenceof either DMSO, W7, or W3. Lineage analysis was conducted for bothlineage-IL7R-ckit+Sca1+(LSK) (FIGS. 46A-46B) andlineage-IL7R-ckit+Sca1+CD48-CD150+ HSCs (FIGS. 47A-47B). 12 dayspost-culturing in the presence of DMSO, W7, or W3. Donor cells obtainedby this ex vivo culturing protocol were transplanted into irradiatedrecipients (FIG. 48A) and donor cell engraftment (FIG. 48B) and lineagecontribution (FIG. 48C) were analyzed 4 weeks post-transplantation of200 starting cell (HSC) equivalents.

What is claimed herein is:
 1. A method of producing an expandedpopulation of hematopoietic stem cells ex vivo, said method comprisingcontacting a population of hematopoietic stem cells with one or moreagents that together exhibit the activities of: a. modulation of histonemethylation; b. inhibition of TGFβ signaling; and c. modulation ofhistone acetylation, wherein the one or more agents are present inamounts that are sufficient to produce an expanded population ofhematopoietic stem cells.
 2. The method of claim 1 wherein thepopulation of hematopoietic stem cells exhibits a hematopoietic stemcell functional potential after two or more days that is greater thanthat of a control population of hematopoietic stem cells cultured underthe same conditions and for the same time as said population ofhematopoietic stem cells but not contacted with said one or more agents.3. The method of claim 1, wherein said agent that modulates histonemethylation is a histone demethylase inhibitor and said agent thatinhibits TGFβ signaling is a TGFβ receptor inhibitor.
 4. The method ofclaim 3, wherein said histone demethylase inhibitor is a LSD1 inhibitor.5. The method of claim 4, wherein said LSD1 inhibitor is LSD1 inhibitorIV RN-1 and said TGFβ receptor inhibitor is ALK5 inhibitor II.
 6. Themethod of claim 4, wherein said LSD1 inhibitor is tranylcypromine andsaid TGFβ receptor inhibitor is ALK5 inhibitor II.
 7. The method ofclaim 1, wherein the one or more agents comprise a combination of agentsselected from the combination of agents of Table 1, Table 2, Table 3,Table 4, Table 5, and Table
 6. 8. The method of claim 1, wherein saidhistone demethylase is LSD1.
 9. The method of claim 8, wherein said oneor more agents comprise a histone demethylase inhibitor selected fromthe group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101,LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, and LSD1 inhibitorI Tranylcypromine.
 10. The method of claim 1, wherein said one or moreagents comprise a compound that inhibits a histone deacetylase areselected from the group consisting of Trichostatin A, valproic acid,butyrylhydroxamic acid, and istodax.
 11. The method of claim 1, whereinsaid one or more agents further comprise a compound that inhibits BMPsignaling.
 12. The method of claim 1, wherein said hematopoietic stemcells are from human cord blood, mobilized peripheral blood, or bonemarrow.
 13. A method of introducing a polynucleotide into a populationof hematopoietic stem cells, said method comprising: a. inserting thepolynucleotide into said population of hematopoietic stem cells; and b.expanding said population of hematopoietic stem cells according to themethod of claim
 1. 14. A method of treating a recipient withhematopoietic stem cells or progeny thereof, said method comprising: a.providing a population of hematopoietic stem cells; b. expanding saidpopulation of hematopoietic stem cells according to the method of claim1; c. optionally differentiating said hematopoietic stem cells intocommon lymphoid progenitor cells, common myeloid progenitor cells,megakaryocyte-erythroid progenitor cells, granulocyte-megakaryocyteprogenitor cells, granulocytes, promyelocytes, neutrophils, eosinophils,basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts,platelet-producing megakaryocytes, platelets, monocytes, macrophages,dendritic cells, microglia, osteoclasts, and lymphocytes, NK cells,B-cells and/or T-cells; and d. introducing the population of expandedhematopoietic stem cells or progeny thereof into said recipient.
 15. Themethod of claim 1, wherein the hematopoietic stem cells are additionallycontacted with a substance that inhibits aryl hydrocarbon receptorsignaling, a prostaglandin, an agonist of Notch signaling, or aninhibitor of SIRT1.
 16. The methods of claim 1, wherein thehematopoietic stem cells are additionally contacted with UM171, ananalog thereof, or a UM171 analog selected from Table
 11. 17. The methodof claim 1, wherein the a population of hematopoietic stem cells isfurther contacted with one or more agents that together exhibit theactivities of: inhibition of p38 signaling; or activation of canonicalWnt signaling or promotion of β-catenin degradation.
 18. The method ofclaim 17, wherein said one or more agents comprise an agent thatinhibits p38 signaling, and wherein said compound is SB203580.
 19. Themethod of claim 17, wherein said one or more agents comprise a compoundthat promotes β-catenin degradation selected from the group consistingof CHIR99021, lithium chloride, BIO, and FGF2.
 20. The method of claim1, wherein the cells are contacted with the one or more agentssimultaneously.
 21. A composition comprising one or more agents thattogether exhibit the activities of: a. modulation of histonemethylation; b. inhibition of TGFβ signaling; and c. modulation ofhistone acetylation.